Systems and methods for genetic sequencing

ABSTRACT

A device including a transparent layer defining a surface exposed to a flow volume and to secure a target polynucleotide template and a detector structure secured to the transparent layer and including a plurality of detectors to detect a signal emitted during nucleotide incorporation along the target polynucleotide template.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a continuation under 35 U.S.C. § 120 of pending U.S.application Ser. No. 14/779,532 filed Sep. 23, 2015, which is a U.S.National Phase Application under 35 U.S.C. § 371 of InternationalApplication No. PCT/US2014/032604 filed Apr. 2, 2014, which claimsbenefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Nos.61/835,428 filed Jun. 14, 2013 and 61/808,105 filed Apr. 3, 2013. Theentire contents of the aforementioned applications are incorporated byreferences herein.

FIELD OF THE DISCLOSURE

This disclosure, in general, relates to systems and methods for geneticsequencing.

BACKGROUND

Increasingly, genetic sequencing is being utilized in research andmedicine. In particular, genetic sequencing is used to characterize,analyze, and manipulate characteristics of plants, for example, toimprove productivity of food crops. Further, genetic sequencing is usedin efforts to classify and characterize animals, including researchinganimal migration and species branching. In another example, geneticsequencing is used in medical research to identify genetic-baseddiseases, classify patient response to medicine or treatment, ordetermine characteristics indicating susceptibility to disease.

While many techniques are available for performing genetic sequencing,conventional techniques utilize extensive sample preparation, increasingcosts associated with labor and consumables and introducing human error.Other techniques utilize large and expensive systems, increasing costsand lab space utilized by such techniques.

SUMMARY

In an embodiment, a system for genetic sequencing includes an integrateddevice having detectors for detecting signals indicating incorporationof a nucleotide during template-dependent nucleic acid synthesis along atarget polynucleotide. The integrated device can further include asurface to which the target polynucleotide can be associated anddefining a wall of a flow volume. The integrated device can furtherinclude an energy propagation layer or one or more filter layers. Thesystem can include a fluidics system and a computational system incommunication with the integrated device.

A method of genetic sequencing can include flowing nucleotides through aflow cell of an integrated device that includes detectors for detectingsignals from the template-dependent incorporation of a complementarynucleotide. In some embodiments, the nucleotide includes an opticallydetectable label, and the method can further include providingexcitation energy to the integrated device, the excitation energy eitherexciting a donor particle or molecule that energizes or excites theoptically detectable moiety. Signals emitted during nucleotideincorporation are detected by the detector, leading to a determinationof base identity for the incorporated nucleotide.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure may be better understood, and its numerousfeatures and advantages made apparent to those skilled in the art byreferencing the accompanying drawings.

FIG. 1 includes an illustration of an exemplary system for geneticsequencing.

FIG. 2 and FIG. 3 include illustrations of exemplary devices forperforming genetic sequencing.

FIG. 4 includes an illustration of an exemplary pixel array.

FIG. 5, FIG. 6, FIG. 7, and FIG. 8 include illustrations of exemplarypixels.

FIG. 9 includes an illustration of an exemplary pixel array.

FIG. 10 and FIG. 11 include illustrations of exemplary pixels.

FIG. 12 includes an illustration of exemplary circuit diagram.

FIG. 13 and FIG. 14 include illustrations of exemplary detectors formedin a substrate.

FIG. 15 includes an illustration of exemplary circuit diagram andsubstrate.

FIG. 16 and FIG. 17 include illustrations of exemplary circuit diagrams.

FIG. 18 and FIG. 19 include illustrations of exemplary chiparchitectures.

FIG. 20 includes a cross-section illustration of exemplary filter layer.

FIG. 21 includes a plan view illustration of an exemplary filter.

FIG. 22 includes a plan view illustration of exemplary excitation energyfilter.

FIG. 23 includes an illustration of exemplary integrated device.

FIG. 24 and FIG. 25 include illustrations of exemplary excitationlayers.

FIG. 26, FIG. 27, and FIG. 28 include illustrations of exemplaryintegrated devices.

FIG. 29, FIG. 30, and FIG. 31 includes illustrations of exemplaryevanescent wave focusing structures.

FIG. 32 includes an illustration of an exemplary device.

FIG. 33 and FIG. 34 include illustrations of exemplary surfaces forsecuring a polynucleotide sample.

FIG. 35, FIG. 36, and FIG. 37 include illustrations of exemplary systemsfor fluorescent detection of nucleotide incorporation.

FIG. 38, FIG. 39, and FIG. 40 include illustrations of sequencingschemes.

FIG. 41 includes a graph illustration of an exemplary signal responseduring sequencing.

FIG. 42 and FIG. 43 include flow diagrams illustrating exemplary methodsfor utilizing an exemplary system.

The use of the same reference symbols in different drawings indicatessimilar or identical items.

DETAILED DESCRIPTION

In an exemplary embodiment, a target polynucleotide is linked to asurface of a device and is exposed to nucleotides, such as labelednucleotides. Signals indicative of nucleotide incorporation are detectedby detectors integral to the device. Exemplary detectors can be pHsensors, heat detectors, photon detectors, or combinations thereof. Thetarget polynucleotide can be linked to the surface directly orindirectly. For example, the target polynucleotide can couple with aprobe bound to the surface or can be captured by an enzyme secured tothe surface. In some embodiments, labels of labeled nucleotidesfluoresce upon incorporation along the target polynucleotide, providinga fluorescent signal to be detected by detectors integral to the device.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, apparatuses and devices, forcontacting a device with an enzymatic reaction, where the enzymaticreaction comprises a nucleic acid sequencing reaction. The nucleic acidsequencing reactions include any type of template-dependentsequence-by-synthesis method, including transient-binding reactions,optically-detectable reactions, energy transfer reactions (e.g., FRET),and dark nucleotide reactions (non-labeled nucleotides). In someembodiments, the device comprises an integrated device having detectorsthat detect signals from a nucleotide incorporation reaction, includingany device described herein.

In an exemplary embodiment, a system includes an integrated device fordetecting signals, e.g., fluorescence signals, indicative of nucleotideincorporation. In some embodiments, when a complementary opticallylabeled nucleotide is hybridized to, or is incorporated along a targetpolynucleotide, the label can emit an optically detectable signal. Thesignal from the label can be detected and used to determine baseidentity of the incorporated nucleotide. The system can include afluidics system for feeding nucleotide solutions to the integrateddevice and can include computational systems for controlling theintegrated device and for gathering data from the integrated device. Theintegrated device can further include a surface proximal to which anucleotide is incorporated, for example, to extend a primercomplementary to a target polynucleotide. The integrated device canfurther include detectors for detecting signals indicative of thenucleotide incorporation. Such signals can be emitted, for example, fromfluorescent labels coupled to nucleotides to be incorporated and canresult from fluorescence resonance energy transfer (FRET)-basedfluorescence. Optionally, the integrated device can further includeenergy propagating layers for transferring energy to donors or afluorescent dye, layers for filtering light to provide selectivity for adesired wavelength, and a lid to define a flow volume through whichnucleotide solutions flow. Layers can include one or more substantiallyplanar structures formed of materials having a desired physical,chemical, or optical property.

In an exemplary method, an integrated device is inserted into a systemand coupled to communicate with a computational system and in fluidcommunication with a fluidics system. A target polynucleotide is appliedat surface locations within the integrated device. Solutions includingnucleotides modified with fluorescent dye are provided through thefluidics system to the integrated device. Fluorescent signals resultingfrom incorporation of nucleotides complementary to a targetpolynucleotide are measured by detectors forming a portion of theintegrated device. Detected fluorescence is accessed by thecomputational device for further analysis.

In an example, FIG. 1 illustrates a system 100 for performing geneticsequencing. The system 100 includes an integrated device 102 including aflow cell 104 in fluid communication with a fluidics system. Thefluidics system includes a plurality of reagent solution containers 114in fluid communication with a valve structure 110, which is in fluidcommunication with a port of the flow cell 104 of the integrated device102. Fluid flowing from the valve system 110 through the flow cell 104passes to a waste port 112. The fluidics system is controlled by a flowcontroller 122 forming part of one or more computational systems 116.The flow controller 122 can selectively apply a reagent solution, suchas a wash solution or one of one or more nucleotide solutions to theflow cell 104 of the integrated device 102. In an example, the integratedevice 102 can incorporate a charge coupling device (CCD), acomplementary metal oxide semiconductor (CMOS), or a digital signalprocessor. In a further example, the integrated device 102 canincorporate a capacitive transimpedence amplifier. In a further example,the integrate device 102 can include a photomultiplier tube.

The integrated device 102 can also be in communication with a controller120 and an analyzer 118 of the computational systems 116. In addition,the computational systems 116 can include other processing systems 124and interfaces 126, such as network interfaces and user interfaces. Thecomputational systems 116 can be integrated into a single unit.Alternatively, the computational systems 116 can be remote from theremainder of the system, operating on a network, cloud, or othergrouping of computational devices. In a further example, the controller120 or other control circuitry can control temperature, wavelengthfluorescent excitation power, pressure or other parameters.

The system 100 can further include an excitation source 106 to provideexcitation energy 108 to the integrated device 102. For example, theexcitation source 106 can provide laser light or electromagnetic energyto one or more layers of the integrated device 102. Such excitationenergy 108 can provide energy to fluorescent dye or energy donors.Alternatively, such energy sources can be integrated into the integrateddevice 102. In a particular example, the controller 120 is incommunication with the excitation device 106 and controls excitation ofthe integrated device 102 to selectively periodically excite donorparticles or a dye, which provides energy to be emitted in response tonucleotide incorporation. In an example, the controller 120 cancommunicate both with the excitation source 106 and the integrateddevice 102 to provide fluorescent lifetime imaging. In another example,the excitation source 106 can interface with the integrated device 102using a fiber optic faceplate. The excitation source 106 can be formedof light emitting diode (LED), such as an organic light emitting diode(OLED), or a LED of specific wavelength, such as a blue LED.

In a particular example, the reagent solutions 114 can include a washsolution, and a solution including four types of modified nucleotides,each type of nucleotide optionally modified with a different labelhaving a different emission spectrum. The nucleotide solution can beapplied to the integrated device 102. As nucleotides are incorporated toextend a primer complementary to the target polynucleotide, signals areemitted in a wavelength spectrum that corresponds with the incorporatednucleotide. The spectrum can be a narrow set of wavelengths or can be aset of wavelengths emitted by a dye or dyes associated with a type ofnucleotide. Detectors within the integrated device 102 detect the signaland provide a series of signals to the analyzer 118 and other processing124 to determine the sequence of one or more nucleotide bases that areincorporated to the extending nucleic acid molecule.

In another example, the reagent solution containers 114 can include awash solution and a plurality of solutions that each includes a type ofdye modified nucleotide, wherein each type of nucleotide is modifiedwith a dye having the same or a different emission spectrum. Thenucleotide solutions can be fed to the integrated device 102sequentially. Fluorescent signals emitted as a result of nucleotideincorporation can be detected by detectors within the integrated device102. Data associated with a series of detections can be provided to theanalyzer 118 and other processing 124 for determining genetic sequenceinformation.

In a particular example, a target polynucleotide is linked, directly orindirectly to a surface. Labeled nucleotides provide a signal uponincorporation that is detected by the integrated device 102 and providedto the analyzer 118. For example, a donor molecule or particle, such asa quantum dot (Qdot) nano-crystal, can be tethered to an enzyme, such asa DNA polymerase, without changing the functional properties of theenzyme. The donor molecule or particle absorbs light from a laserexcitation source (e.g., source 106 or a source integrated with thedevice 102) and conditionally transfers energy to a labeled nucleotidedepending on an incorporation event. Each of four nucleotides isterminally labeled with one of four different organic florescent dyes.Strands of the target polynucleotide are immobilized on a surface whilethe donor molecule or particle labeled enzyme binds to the primer-targetcomplexes. The nucleotides are added to start the synthesis of DNA. Whena nucleotide binds to the enzyme, due to close proximity to the donormolecule or particle, resonance energy is transferred to the nucleotidedye, which then emits its own light, for example, in the 600 nm-800 nmrange. The light is collected and optionally spectrally separated anddetected by one or more detectors associated with the location of theimmobilized target polynucleotide. The label of the labeled nucleotidecan be cleaved when the enzyme incorporates the nucleotide. Since theenergy transfer occurs in the vicinity of the donor molecule orparticle, the signal is spatially confined to the enzyme and thenucleotides in solution are rarely caused to emit light. This spatialseparation of emissions keeps the background noise low. A second signalthat indicates base incorporation comes from a measured decrease inintensity from the donor molecule or particle itself as it transfersenergy to the nucleotide label.

In an exemplary embodiment, various features of the stimulation anddetection system are integrated to increase performance and lower thecost of the system. The system is highly scalable, potentially achievingbillions of simultaneous single molecule DNA sequencing runs on a singlelow cost substrate.

FIG. 2 includes an illustration of exemplary integrated device 200 inwhich detectors are formed within a substrate 202, such as a CMOSsubstrate. Optionally, the substrate 202 is created from a semiconductormaterial in which the excitation source is integrated along with thedetecting elements. A flow volume 224 is defined between a lid 216 and asurface layer 212. Target polynucleotides 220 are coupled, directly orindirectly, to the surface layer 212 in regions defined over detectorsdefined within the layer 202. In particular, the target polynucleotides220 can be captured proximal to specific treated areas of the surface212 or within wells 218 defined by a well structure 214. For example,the target polynucleotide can be bound directly to the surface layer212. In another example, the target polynucleotide can be captured by acomplementary primer that is bound to the surface layer 212. In afurther example, the target polynucleotide can be captured by an enzymebound to the surface layer 212. In some embodiments, the targetpolynucleotide can be bound to a bead. In some embodiments, thesubstrate can be a planar substrate with a bead array or a substratewith wells, with the beads located in the wells. Optionally, the surfacelayer 212 can be formed of a fiber optic plate, a transparent ceramic,such as silica or alumina based materials, or like surfaces. Discretesites on the surface layer 212 to which target polynucleotides arecoupled can correspond with detectors within the substrate 202.

In an example, a functional semiconductor substrate is fabricated usingcomplementary metal oxide semiconductor (CMOS) processing. Variousimplants are used to form photodiodes and transistors specific fordetecting the incorporation events that can occur at very low intensity.When photons from the incorporation event fall onto the CMOS substrate,electron-hole pairs are created. In the case of a p-type substrate,holes are drained into the substrate, while the electrons are confinedin potential wells until they are readout by proper circuitry containingtransistors, electrodes or relevant parasitic structures. Althoughelectron confinement is discussed, all similar principles are applied tohole confinement and readout. In a particular example, electron-holepairs can be created at a depth in the silicon substrate that isdependent on wavelength of the radiation. For example, blue photonscreate electron-hole pairs near the surface of the silicon, while redphotons create a majority of electron-hole pairs deeper than a fewmicrons into the substrate. In an embodiment described in more detailbelow, a single pixel can be created in close proximity to thepolymerase in which the “color” of the light can be differentiated byusing multiple confinement wells at different depths within thesubstrate. For example, the single pixel can include at least twoconfinement wells, such as at least three confinement wells, butgenerally less than 10 confinement wells. The confinement wells can havea depth of not greater than 5 micrometers, such as not greater than 3micrometers. In particular, a shallow confinement well can be centeredaround 1 micrometer depth and a deep confinement well can be centeredaround 2 micrometers depth. The number of electrons captured in eachconfinement well indicates which base is incorporated. The confinementwell for the secondary signal (photons coming directly from the Qdot)can also be designed with selective sensitivity, most likely near thesurface. A well capacity for the secondary signal can be createdspecifically for the parameters of the system. The wavelengths using thehigh levels of sensitivity can be designed to reduce surface states. Ifthe secondary signal is large relative to the colored signals, a surfacediode can be used, which normally has a higher dark current level, butis acceptable for sufficiently large signals. The other confinementwells can be “buried” with pinning implants to eliminate surface defectinduced dark current. A CCD collection electrode may also be combinedalong with photodiode implants, creating a hybrid CCD/photodiode pixel.Such a configuration has advantages when different well capacities areused between the primary and secondary signals.

Optionally, the integrated device 200 includes an energy propagationlayer 210, such as a layer that provides for total internal reflection(TIRF) or that provides for energy propagation creating an evanescentwave proximal to the target polynucleotides 220. In an example, theenergy propagation layer 210 propagates energy, such as photonic energy,along a path that is generally parallel (e.g., TIRF) to the surface 212.In a particular example, an evanescent wave is produced by thepropagation of energy in the energy propagation layer 210. The energypropagation layer 210 can include a transparent layer through whichlight or other electromagnetic energy is transmitted. Such a transparentlayer can be formed of a transparent ceramic, such as silicon dioxide orindium tin oxide, or can be formed of a transparent polymer, such as apolycarbonate or a transparent fluoropolymer. In another example, theenergy propagation layer can be formed of a material conducive tocarrying electromagnetic energy in a plane parallel to the surface layer212. Such materials can include thin conductive layers, such as metallayers. In an alternative example, the surface layer 212 forms theenergy propagating layer and total internal reflection is caused by adifference in the index of refraction between an aqueous solution andthe layer (212 or 210). In a further alternative, an excitation energysource can be disposed above the target polynucleotide 220.

The device 200 can further include a layer 208 to facilitate totalinternal reflection within the energy propagation layer 210. The layer208 can be reflective to wavelengths associated with the excitationenergy propagated through the energy propagation layer 210. In anotherexample, the layer can have an index of refraction different from thatof the energy propagation layer 210 and causing reflection of thepropagating energy at particular incident angles.

In another example, the integrated device 200 can include a layer 206 tofilter excitation energy propagating within the layer 210. Inparticular, the filter layer 206 can include materials that selectivelypermit transmission of wavelengths associated with fluorescent signalsfrom dye of the modified nucleotides, but are at least partially opaqueto wavelengths associated with excitation energy. For example, thefilter layer 206 can include GaAs, polysilicon, CdS, CdSe, or acombination thereof.

In an additional example, the integrated device 200 can include a filterlayer 204 to further filter fluorescent signals resulting fromnucleotide incorporation into component spectra to be detected bydetectors within the layer 202. An exemplary filter layer materialincludes doped silicon dioxide, doped zirconia, or another predominantlytransparent material doped with a coloring agent.

Optionally, crosstalk prevention structures 222 can be provided thatextend through one or more layers towards the energy propagation layer210, defining pixels and preventing crosstalk of fluorescent signalsbetween pixels. The crosstalk prevention structures 222 can extendthrough the filter layers, such as filter layers 204 or 206, andoptionally within the substrate 202.

During operation, solutions including one or more nucleotides modifiedwith fluorescent dyes can flow through the flow volume 224 and may ormay not be incorporated to extend a primer along a target polynucleotide220. Excitation energy can be provided through the energy propagationlayer 210 which excites the fluorescent dye or optionally donorparticles in proximity to fluorescent dye being incorporated along thetarget polynucleotide 220. During incorporation, the fluorescent dye ofmodified nucleotides fluoresces, and such fluorescence is detected bydetectors within the substrate 202. Optionally, excess excitation energyis filtered by filter layer 206 and the fluorescence can be separatedinto different spectrum by the filter layer 204 to be detected bydifferent detectors formed within the substrate 202. While the filterlayer 206 for filtering excitation energy is illustrated as beingfurther from the detector than the filter layer 204, the position ofsuch filter layers can be reversed when both are included within theintegrated device 200. Fluorescent signals from adjacent wells orregions on the surface structure 212 can be blocked by the crosstalkprevention structures 222.

FIG. 3 includes an illustration of another exemplary integrated device300. A layer 302 in which detectors are defined is provided on anopposite side of the flow volume 324 from the surface 312 to whichtarget polynucleotides 320 are proximal. The integrated device 300 canfurther include an energy propagation layer 314 and associatedreflective layer 316. The energy propagation layer 314 can provideevanescent wave energy to facilitate fluorescence of modifiednucleotides being incorporated along the target polynucleotide 320. Thetarget polynucleotide 320 can be coupled, directly or indirectly, tospecific locations on the surface layer 312 or can optionally beconfined by wells 318 defined by a well structure 310, as describedabove. A transparent lid layer 308 can, with the surface layer 312,define the flow volume 324. The integrated device 300 can furtherinclude a filter layer 306 to filter excitation energy and can include afilter layer 304 to filter fluorescent spectrum into various detectionspectrum, as described above. The integrated device 300 can furtherinclude crosstalk prevention structures 322 extending through one ormore of the filter layers 306 or 304 and optionally into the layer 302into which the detectors are formed.

In operation, nucleotide solutions including nucleotides modified withfluorescent dyes flow through the flow volume 324 and may beincorporated into or along a polynucleotide target 320. The dyeassociated with the nucleotide can be energized by a donor particle thatis energized by an evanescent wave or can be energized by the evanescentwave itself. The evanescent wave can be provided by the energypropagation layer 314. Fluorescent signals resulting from theincorporation of the nucleotide along the target polynucleotide 320 canpass through the flow cavity 324 and the lid layer 308 and optionallythrough filters 304 or 306 for filtering excitation energy andseparating fluorescence into various detectable spectrums by thedetectors defined within the layer 302. Fluorescence from adjacenttarget polynucleotides can be prevented from impinging on otherdetectors by the crosstalk prevention structures 322.

The crosstalk prevention structures, such as 222 or 322, can be formedof reflective material or material that is opaque to the wavelength ofexcitation energy or the fluorescence spectra. For example, thecrosstalk prevention structures 222 or 322 can be formed of metal, suchas aluminum, copper, titanium, gold, silver, platinum, or anycombination thereof. In another example, the crosstalk preventionstructures 222 or 322 can be formed of polysilicon or doped polysiliconhaving a thickness sufficient to limit transmission of photons acrossthe structure 222 or 322. When the optical detectors are electronic innature, the crosstalk prevention structures can also be insulative.

As illustrated in FIG. 2 or FIG. 3, layers can be secured to one anotherdirectly or indirectly, such as through intermediate layers or byadhesives. For example, the surface layer 212 is indirectly secured tothe substrate 202, by one or more optional layers, such as filter layers204 or 206 or the propagation layer 210. In another example, thesubstrate 202 can be directly secured to a filter layer 204 by formingthe filter layer 204 on the substrate using semiconductor processingtechniques. In further examples, the different layers can be thermallybonded together, in particular, without intervening adhesive layers.

As illustrated in FIG. 4, detectors defined within a substrate can begrouped as pixels, such as pixels 402. Each pixel 402 can include one ormore detectors arranged adjacent to each other within a horizontal planeor arranged to overlie one another and extending into a substrate. Asillustrated in FIG. 4, pixels can be arranged within defined squares.Alternatively, pixels of other shapes and various orientations can beformed.

For example, FIG. 5 illustrates a top view of an exemplary pixelincluding a first detector 502 and a second detector 504 side-by-side ina plane. Each detector 502 or 504 may be responsive to differentwavelength spectrum resulting from fluorescent signals of different dye.In another example, each of the detectors 502 or 504 can be similar innature detecting a broad spectrum of wavelengths and having differentfilters associated with each detector 502 or 504.

Similarly, FIG. 6 illustrates a top view of a four detector pixelincluding exemplary detectors 602, 604, 606 and 608. As with thedetectors of FIG. 5, the detectors are arranged adjacent each other whenviewed from the top view. The detectors may each be configured to detectdifferent wavelengths spectrum. In another example, the detectors caneach be broad-spectrum detectors associated with different filtersselective to the emission spectrum of particular dye. When using a darknucleotide, the pixel can optionally have fewer than four detectors.

While FIG. 5 and FIG. 6 illustrate rectilinear shaped detectors, thedetectors can alternatively be formed in various shapes. For example,FIG. 7 illustrates an exemplary circular pixel including quarter circleshaped detectors 702, 704, 706, or 708. When viewed from the top view,such detectors are adjacent one another, for example, directly adjacentor separated by an opaque divider or insulator. The detectors 702, 704,706, or 708 can each be configured to detect a different wavelengthspectrum or can be configured to detect a broad spectrum of wavelengthsand be associated with different filters selective to the emissionspectrum of particular dye.

FIG. 8 illustrates a top view of an exemplary circular pixel includingsix detectors 802, 804, 806, 808, 810 and 812. Pixels can be formed toinclude four detectors for detecting four different wavelength spectrumassociated with the fluorescence from four different dyes, eachassociated with a different type of nucleotide. In a further example, afive or six detector system can be defined which further includesdetectors for excitation energy or background noise.

FIG. 9 illustrates an alternative system that includes polygonal pixels902. Such polygonal pixels 902 can include four or more detectors, suchas detectors 904, 906, 908, 910, 912, or 914. The detectors can beselective to a particular spectrum associated with a particular dye orcan detect wavelengths associated with excitation energy or backgroundnoise. Alternatively, additional detectors may be utilized in the aboveconfigurations for enhancing the detection area associated withwavelength spectrum of dye that is more difficult to detect thanfluorescence from other alternative dye. In further examples of theabove pixel configurations, an emitter, such as a light emitting diodecan be incorporated into each pixel.

In an example, one or more layers can be provided that filter specificwavelengths from the emitted signal. In such an example, genericdetectors can be utilized underlying different filters and resulting inthe detection of different wavelengths of light within the variousdetectors. In particular example, layers of various thickness ofmaterial that is semi-opaque to fluorescence emitted by the fluorescentdyes can be utilized to filter fluorescent light before it impingesvarious detectors. For example, as illustrated in FIG. 10, a layer 1002can include detectors 1006, 1008, or 1010. In an example, the layer 1002is a polysilicon layer or gallium arsenide layer in which detectors1006, 1008, and 1010 and associated circuitry are formed. Structures ofdifferent thickness are formed over different detectors and are formedof material that is semi-opaque or provides resistance to photons ofdifferent wavelengths. For example, different thicknesses ofpolysilicon, gallium arsenide or other semi-transparent ceramics orpolymers can be applied over the layer 1002 including the detectors1006, 1008, 1010. For example, the detector 1006 can be free of astructure, where as the detector 1008 can be associated with a structure1012 having a first thickness and the detector 1010 can be associatedwith the structure 1014 having a different thickness. Such structures1012 and 1014 can be embedded within a transparent material, such as asilicon dioxide, zirconium dioxide, indium tin oxide or othertransparent material. Owing to the nature of the penetration of photonsof different wavelengths, wavelengths that are more red in nature canpenetrate the structure 1014, activating the detector 1010, wavelengthsthat are green in nature can penetrate structure 1012 and activate thedetector 1008. In a further example, wavelengths that are more blue innature can activate the detector 1006 while other wavelengths penetratethrough the detector 1006 without activating it. While red, green andblue are described to illustrate the nature of the technique, thetechnique can be extended to other wavelengths by adjusting layerthickness. In such a manner, the nature of the wavelengths that resultin a reading within a detector 1006, 1008, or 1010 is a function of thethickness of the associated structure, such as structure 1012 orstructure 1014.

In a particular example, the thickness of the polysilicon is adjustedfor each color. For example, a thick polysilicon layer absorbs blue andgreen but may allow the majority of red photons to pass through. A pixelwith no polysilicon layer may allow all colors to pass through. In thesimplest form, two pixels, one containing a polysilicon layer and onewithout a polysilicon layer, can be used to differentiate four differentnucleotide incorporation events. These absorption layers can beimplemented at the silicon substrate or after backend metal layers areprocessed along with the dielectric layers. Employing absorption layersin both the frontend of the process and the backend of the process givesadditional options for selectivity.

In an alternative example, illustrated in FIG. 11, a cross-section of asemiconducting structure 1102 illustrates detectors 1104, 1106, 1108disposed over one another. Fluorescent signals of different wavelengthspenetrate the cross-section of the semiconductor structure 1102 todiffering levels resulting in the activation of the detectors 1104,1106, or 1108 depending upon the nature of the wavelength of the signalpassing through the semiconductor material 1102. In particular, longerwavelength spectrum activate deeper detectors where as shorterwavelength spectrum are prevented from passing further into thesemiconductor material and activate more shallow detectors. Such isparticularly the case for features formed within polysilicon or similarsemiconductor materials. Alternatively, a combination of the structuresof FIG. 10 and FIG. 11 can be used to detect multiple wavelengths usingless area.

FIG. 12 includes an illustration of exemplary circuitry for detectingcharge within two levels of a CMOS device. Each diode 1202 or 1204 isfully depleted during charge transfer. A pixel first transfers theshallow charge from 1202 by activating TX1 (1206). The floatingdiffusion is reset with RT (1210). Correlated double sampling is used toremove thermal noise. The deep charge is transferred by activating theTX2 (1208). Three samples can be used for removal of thermal noise. Sucha circuit configuration can, for example, be used in conjunction withexemplary device cross-sections illustrated in FIG. 13 and FIG. 14.

FIG. 13 includes an illustration of exemplary double pinned photodiode.After polysilicon 1410 deposition, a deep implant 1408 is formed,patterned and annealed. The deep implant 1408 can also be formed at thebeginning of the CMOS in processes using lower energy. Compensatingimplant 1406 reduces the size of the deep implant and creates a barrierbetween the deep implant 1408 and the shallow implant 1404. The shallowimplant 1404 is self aligned to the second electrode 1418. After siliconnitride gate spacers are formed, a p-type layer 1402 is implanted andannealed.

In an alternative example, the deep implant 1508 is not self alignedwith the polysilicon and is made with a single energy and dose. Thenative p-epitaxy region 1510 forms the barrier between the shallowjunction and a narrow junction. The pixel is read with the shallowjunction first being fully depleted before the deep junction charge istransferred. A p-type implant 1506 at an electrode 1518 creates asufficient barrier to prevent charge transfer from the deep junction tothe floating diffusion 1512. Since the epitaxy region 1510 is lightlydoped, it is depleted easily to the depth of the deep implant 1508. Whenthe electrode 1516 is switched to high potential, the electrons in thedeep implant are transferred to the floating diffusion 1514.Consequently electrons in the shallow junction 1504 may also betransferred during the cycle. For this reason, the shallow junctionshould be empty first. Alternatively, the shallow junction can be pulledback sufficient distance from the electrode 1516, which may decrease thequantum efficiency of the shallow junction.

FIG. 15 illustrates a further exemplary construction in which regions1602, 1604, or 1606 each include associated circuitry for selectivelydetecting charge within the layers and thus providing spectral selectionbased on the depth of the layers within the CMOS device. In an example,each well can be reset to a type specific charge when reset is selected.In particular, p-type wells can be reset to Vp, or n-type wells can bereset to Vn. In response to a row select, each color can be read througha separate column channel.

FIG. 16 illustrates an alternative circuitry utilizing a single pinneddiode. Such architecture provides a low noise. The photodiode can becharged by activating TX2 when TX1 is off and can be read by activatingTX1 when TX2 is off. Charge from the photodiode is transferred to thenode and activates the transistor associated with SF. A resulting signalis provided to the column bus when the associated row is selected. Thecapacitor C_(fd) can be reset by activating RST.

FIG. 17 includes an illustration of an exemplary structure forextracting data from the system. Data enters from a column bus andpassed through a low gain or a high gain filter. The data is digitizedand stored in a digital memory for output. When using the high gainpath, the system can deliver as much as 200 frames per second (fps) ormore (e.g., 120 fps to 1028 fps) and provides a dynamic range of 8 bitsor higher (e.g., 8 bits to 32 bits).

FIG. 18 and FIG. 19 illustrated chip architectures for use by theintegrated device and the circuitry of the integrated device. Eacharchitecture includes more than one pixel array, such as first andsecond pixel arrays, each associated with column amplifiers and columnanalog-to-digital converters. In addition, the architecture includescolumn decoders. By splitting the pixel array into one or more units,and accessing the units separately more data can be collected throughthe array. In a particular example, the multi-array combination caninclude as many as 300 M or more detectors having a pixel size of 3 μmor less, such as less than 2 μm. Pixel arrays can be arranged inmultiples of 2160×2560 or higher aspect ratios. Further, such CMOSintegrated devices can access the data at greater than 200 frames persecond and 8 bits and an RN of 2e− at 200 fps. When using four detectorsper pixel, the chip may include at least 75 million pixels with a dataflow rate of 270 GB per hour.

Since the incorporation events occur asynchronously over the course ofthe DNA synthesis, a high frame rate is used to capture incorporationevents. It is advantageous to reduce the data at each incorporation siteto build high density systems. The data at each site can be limited tothe color of the nucleotide and whether or not the event is a trueincorporation, utilizing 3 bits. For example:

000—no incorporation001—no incorporation010—no incorporation011—no incorporation100—Red labeled nucleotide101—Orange labeled nucleotide110—green labeled nucleotide111—yellow labeled nucleotide

A mapping of these bits to the depth of the confined photo charge withintwo confinement wells can be used with the confinement wellsillustrated, for example, in FIG. 13 or FIG. 14. To provide three bitsfrom each incorporation site, comparison thresholds may be implementedwithin the circuitry local to the incorporation site. The bit thatdetermines whether an incorporation has occurred or not can come fromthe secondary signal or from a decision rule based on the totalintensity within a pixel. Since the photodiodes at each incorporationsite act as a memory, the required frame rate can be fast enough thattwo incorporation events cannot occur within a frame interval.

In addition or alternatively, the system can include filters that filterfluorescence into various wavelengths to be provided to differentdetectors. Although the spectral content of the primary signal can beseparated by confining the photogenerated electrons at multiple depthswithin silicon, other methods may also be effective. Pigment or dyebased organic color filters can be patterned above each pixel usingphotolithography methods. At least two different colors can be used todifferentiate between four labeled nucleotides. Absorption filtering mayalso be effective by fabricating polysilicon layers above each pixel asshown above in FIG. 10. Regarding patterned color filters, FIG. 20illustrates a cross-section of a device including a CMOS device layer2102 including detectors 2106. A filter layer 2104 includes dopedregions 2108 and 2110, each doped with a different dopant so that theregion 2108 or 2110 are selective for a different wavelength spectrum(λ₁ or λ₂). In such a manner, similar detectors 2106 can be utilized todetect different wavelength spectrum based on the filtering ofassociated regions 2108 or 2110.

FIG. 21 illustrates a top view of a similar system. Within the filterlayer 2200, regions 2202, 2204, 2206, or 2208 can filter differentwavelength spectrum λ₁, λ₂, λ₃, or λ₄. Detectors residing underneathsuch a filter 2200 can detect different wavelength spectrum.

An additional filter layer can be provided that filters excitationenergy but permits the wavelength spectra emitted from fluorescent dyeto pass through the filter. For example, FIG. 22 illustrates a top viewof a set of filter regions within a filter layer 2300. Each filterregion 2302 permits passage of the wavelength spectrum λ₁-λ₄ associatedwith fluorescence signals from dye while being spectrally selectiveagainst wavelengths associated with excitation energy. While the filterlayer 2300 is illustrated as being subdivided into regions 2302 that arespectrally selective, a uniform filter layer can be provided that isspectrally selective for the wavelengths emitted by differentfluorescent dye associated with nucleotide incorporation.

Optionally, a microlens configuration can be utilized to focus emittedfluorescence onto detectors. For example, the integrated device caninclude a layer incorporating microlenses. As illustrated in FIG. 23, aCMOS layer 2402 that includes detectors 2414 can be situated on anopposite side of a microlens layer 2406 from regions 2412 in whichfluorescence occurs as a result of nucleotide incorporation. Forexample, the integrated device can include an energy propagation layer2410. A microlens layer 2406 can be disposed below the energypropagation layer 2410 and filter layers 2404 can be disposed betweenthe microlens layer 2406 and the detector layer 2402. The microlenslayer 2406 can include microlenses 2408 aligned between the regions 2412and the detectors within the detector layer 2402.

Energy propagation layers that provide total internal reflection ofexcitation energy can be used to provide an evanescent wave within thesolution. Such evanescent waves can energize donors which donate energyto fluorescent dye or can energize the fluorescent dye itself. In aparticular example, excitation light can be received from an externalsource. For example, as illustrated in FIG. 24, energy propagation layer2502 can be positioned between two reflective layers 2504 and 2506. Suchlayers 2504 or 2506 can act as reflective layers based on a differencein refraction index relative to the energy propagation layer 2502.Alternatively, the layer 2504 may be absent and reflection can resultfrom a difference in refractive index with an aqueous solution. Anexternal source 2510 provides light through a surface 2508 whichundergoes total internal reflection. Optionally, a mirrored surface 2512is provided which reflects propagating light, further containing thelight within the layer 2502 and preventing the light from exiting theintegrated device. The propagation of the light 2510 within the layer2502 creates an evanescent wave that energizes donors or fluorescent dyein proximity to the layer 2504.

In another example illustrated in FIG. 25, the energy propagation layer2602 is optionally positioned between the reflective layers 2604 and2606. External excitation energy 2610 is applied to a refractive grating2608, altering the path of the light to angles that result in totalinternal reflection within the layer 2602. As with FIG. 24, optionalmirrored surface 2612 can prevent the light from exiting the energypropagation layer 2602.

In a further example, an energy source, such as a light emitting diode(LED) or other energy source, can be integrated within the device. Forexample, as illustrated FIG. 26, the integrated device 2702 can includean energy propagation layer 2704. An energy source, such as a LED device2706, emits energy 2708 which is reflected or refracted based on astructure 2710 and propagates through the energy propagation layer 2704.The structure 2710 can be a refractive grating, reflective surface, orother structures for facilitating total internal reflection within theenergy propagation layer 2704. Instead of using an external lightsource, the CMOS substrate, if advantageous, can be designed to emit itsown light source.

In a particular example, light emitting diodes (LEDs) are created ateach pixel location working on the principle of hot carrier directrecombination. The light source is modulated in conjunction with thereadout of the photodiodes to tune the system for response. Since thelight source can be bound to the silicon substrate, and local to theincorporation site, the donor molecule or particle may be removed,because the light source interacts with the labeled nucleotides that arebound to the polymerase during the incorporation, keeping the backgroundnoise low, while simplifying the system. The light source can beconstantly modulated while the photodiodes are readout during theoff-cycles. If a label lights up and is detected by the pixels at theincorporation site, it most likely comes from a nucleotide that isincorporated into the DNA. Therefore, if the polymerase is bound to thepixel and the light source is local to every pixel, then direct stimulusof the labeled nucleotide can be used and effectively detected.

In a further alternative illustrated in FIG. 27, an integrated device2802 can include a via or opening 2806 through which energy 2808 can betransmitted. Alternatively, the via or opening 2806 can be filled with atransparent material. The energy can be refracted or reflected bystructure 2810 causing total internal reflection within the energypropagation layer 2804. In an example, the structure 2810 includes arefraction grating or a reflective surface. In such an example, aninterface 2812 interfacing with the device 2802 can include a lightsource 2814. Such a light source can be an LED built-in to the interface2810 or can be an optical fiber or light pipe aligned in the interfacewith the via 2806 of the device 2802 to provide light or energy 2808 tothe energy propagation layer 2804. In particular, the interface 2812 canbe formed as part of the system, for example, illustrated in FIG. 1,which accepts a sequencing device incorporating the integrated device2802.

In a further alternative, excitation energy can be provided from anexternal source directly to the donor particles or dye withoutpropagating to a detector. For example, FIG. 28 illustrates an exemplarydevice in which excitation energy 2908 is provided to the surface layer2906. Such excitation energy is prevented from impinging detectorswithin the CMOS layer 2902 by an interference filter including multiplelayers of material having different indexes of refraction. For example,such an interference filter 2904 can include alternating layers ofsilicon dioxide and silicon nitride or other similar materials. Thethickness of such alternating layers can provide reliable selectivityfor fluorescent signal wavelengths and filter wavelengths associatedwith excitation energy. For example, alternating layers of silicondioxide and silicon nitride can be selectively formed to permittransmission of fluorescent light associated with signals resulting fromnucleotide incorporation while preventing transmission of excitationlight. In such a way, excitation light can be provided from an externalsource without an energy propagation layer while still being preventedfrom interfering with detection of the fluorescent emissions.

In another example, when a wave or energy propagation layer is utilized,additional structures can be formed proximal to the energy propagationlayer to enhance the evanescent wave within particular regions. Forexample, conductive structures can be formed that provide edges andpoints configured to enhance or concentrate such evanescent waves withinsmall regions. As illustrated in FIG. 29, structures 3002 can be formedhaving points converging on regions 3004. Such regions when positionedabove an energy propagating layer provide an enhanced evanescent wavefor exciting donor particles or molecules or dye molecules. Similarlyother patterns can be formed, such as the pattern illustrated in FIG. 30including squares 3102 defining proximity regions 3104 or as illustratedin FIG. 31 including polygons 3202 defining regions 3204 that have anenhanced evanescent wave.

Many types of florescent labels may be used and some may have advantagesover others depending on the stimulator that is integrated within thesilicon substrate. For example, a local RF signal may be generated ateach incorporation site. The RF power may be absorbed by the label andemitted as visible light sensed by the detection pixels.

A target polynucleotide can be localized, linked or otherwise secured toa surface such as through hybridization to a complementary primer, orcan be captured by an enzyme secured to the surface. The surface can beformed of transparent or semitransparent material permitting thetransmission of fluorescent emission to a detector. In an example, theflow of target polynucleotides into regions to be detected can becontrolled by electromagnetic potential or charge. In an exampleillustrated in FIG. 32, the device includes a transparent electrode 3304overlying other layers 3302 and defining a surface in proximity to whichnucleotides can be incorporated along a target polynucleotide. Forexample, the electrode layer 3304 can be formed of indium tin oxide(ITO), flouring doped tin oxide, doped zinc oxide (e.g., aluminum dopedzinc oxide), poly(3,4-ethylenedioxythiophene) (PEDOT) and similarpolymers, carbon nanotube networks, grapheme, or combinations thereof.In an example, an electrode of opposite charge can be formed within alayer 3310 defining a flow volume over the system. A potentialdifference between the electrodes 3304 and the electrode 3310 can drivepolynucleotides into a flow region 3312 associated with a detector. Inanother example, an electrode 3308 can be defined within a wellstructure. The electrodes 3308 can be surrounded by dielectric layer3306. Such an electrode 3308 can, for example, be formed of a dopedpolysilicon which is oxidized on an outer surface to provide theinsulating layer 3306. Such an electrode structure 3308 can be utilizedalong with the electrode 3304 or the electrode 3310 to drivepolynucleotides into a well structure or region 3312 or to stretch orcompress such polynucleotides.

As illustrated in FIG. 33, wells can be defined over a surface 3402using a well structure 3404. In particular, the well structure 3404 canbe formed of material that assists with total internal reflection in thepropagation layer. For example, the well structure 3404 can be formed ofa polymeric material having an index of refraction similar to that ofwater. In an example, the polymeric material includes Cytop®. In anotherexample, the well structure 3404 can be formed of a material that isreflective, such as a metallic material including aluminum, copper,titanium, gold, silver, platinum, or a combination thereof.

In another example, pad regions can be formed on a surface to whichpolynucleotides or enzymes can be selectively attached. As illustratedin FIG. 34, pads 3504 can be applied over a layer 3502. In between thepads 3504 can be a polymer material 3506 that prevents attachment ofpolynucleotide species or enzymes. For example, the pads 3504 can beformed of titanium, zirconium, gold, or other materials. In a furtherexample, the pads 3504 can be formed of metallic surfaces, such as gold,silicon, copper, titanium, and aluminum; metal oxides, such as siliconoxide, titanium oxide, and iron oxide; plastics, such as polystyrene,and polyethylene; zeolites, or other materials. In an example, thepolymer includes polyethylene glycol.

Depending on the nature the system, the template polynucleotide can belinked (e.g., covalently or non-covalently) to the surface.Alternatively, an enzyme (e.g., a polymerase or other template-bindingenzyme) can be tethered to the surface and can capture polynucleotides.In FRET-based systems, the enzyme can be associated with a donorparticle. For example, as illustrated in FIG. 35, the templatepolynucleotide 3606 can be attached to a surface 3602 within wellsdefined by a well structure 3604. A polymerase enzyme 3608 can accessthe template polynucleotide 3606 and incorporate fluorescent dyemodified nucleotides 3610 into a nascent nucleic acid molecule (e.g., anextending primer) in a template-dependent fashion. In another exampleillustrated in FIG. 36, the enzyme 3708 can be attached to a donorparticle or molecule 3710. When incorporating a dye modified nucleotide3712 complementary to a corresponding nucleotide within the templatepolynucleotide 3706, the donor particle or molecule can provide energyto the dye of the dye modified nucleotide 3712 causing fluorescence.

In a further example illustrated in FIG. 37, an enzyme 3808 andassociated donor particle or molecule 3806 can be tethered to a surface3802 within a well defined within the structure 3804. Targetpolynucleotides 3810 can be bound by the enzyme 3808, and can serve astemplates for incorporation of dye modified nucleotides 3812 by theenzyme 3808. When incorporated, the dye of the dye modified nucleotide3812 can fluoresce, providing an indication of the incorporation.

Surface binding of the template polynucleotide or of the enzyme can befacilitated using a molecular recognition layer bound to the surface.The surfaces to which the molecular recognition layer is bound can betreated with a layer of chemicals prior to attaching probes to enhancethe binding or to inhibit non-specific binding during use. For example,glass surfaces can be coated with self-assembled monolayer (SAM)coatings, such as coatings of as aminoalkyl silanes, or of polymericmaterials, such as acrylamide and proteins.

Probes can be attached covalently. A number of different chemicalsurface modifiers can be added to the surface to attach the probes.Examples of chemical surface modifiers include N-hydroxy succinimide(NHS) groups, amines, aldehydes, epoxides, carboxyl groups, hydroxylgroups, hydrazides, hydrophobic groups, membranes, maleimides, biotin,streptavidin, thiol groups, nickel chelates, photoreactive groups, borongroups, thioesters, cysteines, disulfide groups, alkyl and acyl halidegroups, glutathiones, maltoses, azides, phosphates, and phosphines.

In some embodiments, surfaces that are reactive to probes comprisingamines are used. Examples of such surfaces include NETS-esters,aldehyde, epoxide, acyl halide, and thio-ester. Most proteins, peptides,glycopeptides, etc. have free amine groups, which react with suchsurfaces to link them covalently to these surfaces. Nucleic acid probeswith internal or terminal amine groups can also be synthesized. Thus,nucleic acids can be bound (e.g., covalently or non-covalently) tosurfaces using similar chemistries.

The surfaces to which the probes are bound need not be reactive towardsamines, but can be easily converted into amine-reactive surfaces withcoatings. Examples of coatings include amine coatings (which can bereacted with bis-NHS cross-linkers and other reagents), thiol coatings(which can be reacted with maleimide-NHS cross-linkers, etc.), goldcoatings (which can be reacted with NETS-thiol cross linkers, etc.),streptavidin coatings (which can be reacted with bis-NHS cross-linkers,maleimide-NHS cross-linkers, biotin-NHS cross-linkers, etc.), and BSAcoatings (which can be reacted with bis-NHS cross-linkers, maleimide-NHScross-linkers, etc.). Alternatively, the probes, rather than the opensurface, can be reacted with specific chemical modifiers to make themreactive to the respective surfaces.

A number of other multi-functional cross-linking agents can be used toconvert the chemical reactivity of one kind of surface to another. Thesegroups can be bifunctional, tri-functional, tetra-functional, and so on.They can also be homo-functional or hetero-functional. An example of abi-functional cross-linker is X—Y—Z, where X and Z are two reactivegroups, and Y is a connecting linker. Further, if X and Z are the samegroup, such as NETS-esters, the resulting cross-linker, NHS—Y—NHS, is ahomo-bi-functional cross-linker and would connect an amine surface withan amine-group containing molecule. If X is NETS-ester and Z is amaleimide group, the resulting cross-linker, NHS-Y-maleimide, is ahetero-bi-functional cross-linker and would link an amine surface (or athiol surface) with a thio-group (or amino-group) containing probe.Cross-linkers with a number of different functional groups are widelyavailable. Examples of such functional groups include NETS-esters,thio-esters, alkyl halides, acyl halides (e.g., iodoacetamide), thiols,amines, cysteines, histidines, di-sulfides, maleimide, cis-diols,boronic acid, hydroxamic acid, azides, hydrazines, phosphines,photoreactive groups (e.g., anthraquinone, benzophenone), acrylamide(e.g., acrydite), affinity groups (e.g., biotin, streptavidin, maltose,maltose binding protein, glutathione, glutathione-S-transferase),aldehydes, ketones, carboxylic acids, phosphates, hydrophobic groups(e.g., phenyl, cholesterol), etc. Such cross-linkers can be reacted withthe surface or with the probes or with both, in order to conjugate aprobe to a surface. Other alternatives include thiol reactive surfacessuch as acrydite, maleimide, acyl halide and thio-ester surfaces.

Such surfaces can covalently link proteins, peptides, glycopeptides,etc., via a (usually present) thiol group. Nucleic acid probescontaining pendant thiol-groups can also be easily synthesized.

Alternatively, one can modify surfaces with molecules such aspolyethylene glycol (PEG), e.g. PEGs of mixed lengths. Other surfacemodification alternatives (such as photo-crosslinkable surfaces andthermally cross-linkable surfaces) can be used.

The examples of FIG. 35, FIG. 36, or FIG. 37 illustrate moleculessecured to a surface within a well. Alternatively, one or more copies ofthe target polynucleotide can be secured to a bead or particle. The beador particle including the target polynucleotide can be disposed over apixel. In a particular example, synchronous sequencing can be performedusing one nucleotide at a time. In such an example, alternative dyes,such as solvatochromatic dyes or dyes sensitive to pH, can be used toprovide an optical signal indicating incorporation of a type ofnucleotide. The bead or particle may or may not be placed within a well.

In another example, beads or particles can be used to deposit capturemolecules at locations, limiting depositions over a pixel by sizeexclusion based on the size of the bead. Such limited deposition can beused with the device of FIG. 34, for example.

In another example, the length of a signal indicative of a nucleotidethat is next in the sequence can be extended by capturing the nucleotideand causing it to emit the signal while preventing incorporation of thenucleotide. In an example, an inhibiting agent can prevent incorporationof the modified nucleotide. An exemplary inhibiting agent includesdivalent metal ions, such as calcium, scandium, titanium, vanadium,chromium, iron, cobalt, nickel, copper, zinc, gallium, germanium,arsenic, and selenium ions. In another example, functionality on thenucleotide can prevent incorporation of the modified nucleotide.

In either case, the enzyme can capture the next complementarynucleotide. Modified functionality of the nucleotide can emit a signal,such as a fluorescent signal. As a result of inhibiting theincorporation of the modified nucleotide, the length of the signal canbe extended. Subsequently, a complementary nucleotide can beincorporated along the sequence. For example, the inhibiting agent canbe washed away, permitting incorporation of the modified nucleotide. Inanother example, a complementary nucleotide modified with functionalityto prevent incorporation can be washed away and replaced with a similarmodified nucleotide having functionality that permits incorporation.

In an example, modified nucleotides can be provided sequentially. Inanother example, the modified nucleotides can be provided in groups oftwo, three, or four types of nucleotides. For example, when nucleotidesare supplied separately and sequentially, the functionalities modifyingthe nucleotides can provide a similar signal, such as a similarwavelength of emission. Such signals, when the modified nucleotides areincorporated, are separated by time allowing identification of thenucleotide.

For example, as illustrated in FIG. 38, a target polynucleotide can becaptured over a sequence detecting device, as illustrated at 4402. Asillustrated at 4404, a type of nucleotide can be provided along with aninhibiting agent, such as calcium ions. The complementary nucleotide(e.g., T) is captured by the enzyme 4420 and caused to fluoresce in anexemplary FRET-based system 4422. When the inhibiting agent is washedfrom solution as illustrated at 4406, the T nucleotide is permitted toincorporate, cleaving the emitting functional group. Other types ofmodified nucleotides can be fed to solution along with inhibiting agent,as illustrated at 4408. When the inhibiting agent is washed solution, asillustrated at 4410, the other non-complementary types of nucleotidesfail to emit a signal or be incorporated. Subsequently, as illustratedin 4412, a complementary modified nucleotide is captured by the enzymein the presence of an inhibiting agent and emits a signal. Even when thesequence includes an adjacent nucleotide of the same type, a singlemodified nucleotide is captured. As illustrated at 4414, when theinhibiting agent is removed, the complementary modified nucleotide canbe incorporated. When in the presence of a homopolymer or adjacentnucleotides of the same type, the modified nucleotide can beincorporated more than once, emitting more than one signal or a signalhaving greater amplitude. As illustrated at 4416 and 4418, subsequentmodified nucleotides can be provided that are not complementary to thetarget sequence. Such non-complementary modified nucleotides do notfluoresce and are not incorporated even in the absence of the inhibitingagent. As a result of delayed incorporation, the signal length is longerand provides for improved detection.

Alternatively, each type of nucleotide (e.g., A, T, C, or G) can bemodified to emit a different signal, such as a signal of a differentwavelength. For example, when two types of modified nucleotides flowtogether through a flow cell, each of the two types of modifiednucleotides can be modified with a functionality that emits a differentwavelength from the other type of modified nucleotide. As such, thenucleotide being incorporated can be identified based on the differentwavelength of emissions, for example. In an example, when a modifiedA-type nucleotide is provided simultaneously with a modified T-typenucleotide, the modified A-type nucleotide can be modified to provide adistinctly different wavelength from the modified T-type nucleotide.Similarly, when C and G-type modified nucleotides are supplied together,each type of modified nucleotide can include functionality that emits ata different wavelength from the other type of nucleotide.

In a further example, four types of modified nucleotide can be providedin solution simultaneously. In such an example, each of the modifiedtypes of modified nucleotides can emit different signals, such as thatat different wavelengths. Alternatively, one type of nucleotide (i.e., adark nucleotide) of the four may not be modified to emit a signal. Insuch an example, the inhibiting agent can be intermittently fed to theflow volume to inhibit incorporation and increase the signal length ofthe nucleotide to be incorporated. When the inhibiting agent is removedfrom the chamber, incorporation can proceed. In another example, oneincorporatable modified nucleotide can be fed to the flow volumesimultaneously with three other unincorporatable modified nucleotides.Solutions including a select incorporatable modified nucleotide (e.g.,A, T, C or G) can be fed simultaneously to the flow volume with threeother unincorporatable types of modified nucleotides. Such solutions canbe fed sequentially or one after the other.

In another example illustrated in FIG. 39, a target polynucleotide ortemplate is captured, as illustrated at 3902. Modified nucleotides maybe provided in pairs. Each modified nucleotide of the pair emits adifferent signal, such as at different wavelengths. As such,incorporation of one type of modified nucleotide relative to the othertype of nucleotide can be identified based on the emitted signal. Asillustrated at 3904, the complementary nucleotide (e.g., T) is capturedand caused to fluoresce. The capture can be performed in the presence ofan inhibiting agent. Subsequently, the complementary nucleotide (e.g.,T) is incorporated (e.g., upon removal of the inhibiting agent), whileanother type of modified nucleotide (e.g., A) is not captured and doesnot fluorescence, as illustrated at 3906. As illustrated at 3908, twoother types of modified nucleotides can be provided, such as, forexample, C-type and G-type modified nucleotides. The complementarymodified nucleotide (e.g., G) can be captured and caused to fluoresce,followed by incorporation, as illustrated at 3910. Capturing can beperformed in the presence of an inhibiting agent. In the presence of ahomopolymer section or a section including adjacent nucleotides of thesame type, the complementary modified nucleotide is incorporated morethan once, providing more than one signal or a higher amplitude signaland leaving the non-complementary nucleotide in solution withoutfluorescing or being incorporated. For example, incorporation along ahomopolymer section can provide a scaled brightness indicative of thenumber of repeats within the homopolymer section. In such an example,the polymerase captures the correct base, causing extended signalemission when in the presence of an inhibiting agent. Alternatively, themethod can be performed in the presence of four types of modifiednucleotides, each emitting a distinct signal.

In a further example illustrated in FIG. 40, a mixture of modifiednucleotides can be provided simultaneously in which one of the types ofnucleotide is able to be incorporated while the remaining types ofnucleotides are modified to prevent incorporation. As such,complementary incorporatable nucleotides can be incorporated, whilesubsequent complementary unincorporatable nucleotides can temporarilyemit a signal, providing a preliminary indication as to which modifiednucleotide is complementary to the adjacent nucleotide in the sequence.As illustrated at 4002, for example, the complementary T-type nucleotidecan be captured and fluoresce. Optionally, such capture can be performedin the presence of an inhibiting agent. As illustrated at 4004, thecomplementary nucleotide is incorporated releasing the modifiedflorescent functionality. The next complementary nucleotide can becaptured by the enzyme and caused to fluoresce. But, such a nextcomplementary nucleotide can be modified to prevent incorporation. Assuch, the unincorporatable modified nucleotide (e.g., G) emits a signalindicative of which nucleotide is complementary to the next position inthe sequence, but is not incorporated. FIG. 41 illustrates an exemplarysignals resulting from the incorporation of a nucleotide and the captureof an unincorporated nucleotide. For example, the first capturedincorporatable nucleotide (e.g., T) emits a short signal, as illustratedat 4102. A subsequently captured unincorporatable modified nucleotide(e.g., G) emits a long signal 4104 indicative of its being complementaryto the sequence, yet is unable to be incorporated. As the solutions arewashed through the system, such an unincorporated nucleotide providespreliminary indication as to which nucleotide is next in the sequence,which is confirmed when a subsequent solution including anincorporatable modified nucleotide of the same type is fed to the systemand emits a signal. Alternatively, more than one incorporatable modifiednucleotide can be included in a solution with one or moreunincorporatable modified nucleotides.

Modified nucleotides can be labeled or modified to provide the abilityto provide a signal in response to incorporation and optionally, toadjust whether the nucleotide or analog thereof can be incorporated.

In one embodiment, the labeled nucleotide can include 3-10 or morephosphate groups. In an example, the labeled nucleotide can beadenosine, guanosine, cytidine, thymidine or uridine, or any other typeof labeled nucleotide. In another example, the label can be an energytransfer acceptor reporter moiety. In particular, the label can be afluorescent dye. The polymerase can be contacted with more than one typeof labeled nucleotide (e.g., A, G, C, or T/U, or others). Each type oflabeled nucleotide can be operably linked to a different reporter moietyto permit nucleotide identity. Each type of labeled nucleotide can beoperably linked to one type of reporter moiety. In an example, thelabeled nucleotides are operably linked at the terminal phosphate groupwith a reporter moiety. In another example, the labeled nucleotides areoperably linked at the base moiety with a reporter moiety. In anotherembodiment, the labeled nucleotide can be a non-incorporatablenucleotide. The non-incorporatable nucleotide can bind to the polymeraseand template nucleic acid molecule which is base-paired to apolymerization initiation site, in a template-dependent manner, but doesnot incorporate. Different types of labeled nucleotides can be employedin the method for detecting the presence of a transiently-boundnucleotide in order to determine the frequency, duration, or intensity,of a transiently-bound nucleotide. For example, a comparison can be madebetween the frequency/duration/intensity of transiently-boundcomplementary and non-complementary nucleotides. Typically, for directexcitation of the reporter moiety, the length of the transient bindingtime of a complementary nucleotide can be longer or more frequentcompared to that of a non-complementary nucleotide. Typically, forFRET-based excitation and detection of the reporter moieties, thetransient binding time of a complementary nucleotide can be of longerduration compared to that of a non-complementary nucleotide.

In one embodiment, the polymerase can be operably linked to an energytransfer donor (e.g., fluorescent dye or nanoparticle). In an example,the labeled nucleotide comprises an energy transfer acceptor moiety(e.g., fluorescent dye). For example, the energy transfer donor andacceptor can be a FRET pair. The signal (or change in the signal) fromthe energy transfer donor or acceptor can be used to detect the presenceof the transiently-bound nucleotide. In a particular example, the signalemitted by the transiently-bound nucleotide can be a FRET signal.

In one embodiment, the excitation source can be electromagneticradiation. The excitation source can be a laser. The signal, or thechange in the signal, can be optically detectable. In an example, thepolymerase has an active site. The active site can beenzymatically-active. The labeled nucleotide can bind the active site,thereby bringing the polymerase and labeled nucleotide in closeproximity with each other. The polymerase may be labeled or unlabeled.In one embodiment, the signal or change in the signal can be afluorescent signal resulting from direct excitation of the label whichis operably linked to the transiently-bound labeled nucleotide or to thelabeled polymerase. In one embodiment, the energy transfer donor oracceptor moieties can fluoresce in response to direct excitation. Thesefluorescence responses can be a signal or change in a signal. In anexample, the energy transfer acceptor moiety can fluoresce in responseto energy transferred from a proximal excited energy transfer donormoiety. These fluorescence responses can be a signal or change in asignal. The proximal distance between the donor and acceptor moietiesthat accommodates energy transfer can be dependent upon the particulardonor/acceptor pair. The proximal distance between the donor andacceptor moieties can be about 1-20 nm, or about 1-10 nm, or about 1-5nm, or about 5-10 nm. The energy transfer signal generated by proximityof the donor moiety to the acceptor moiety can remain unchanged. Inanother example, the energy transfer signal generated by proximity ofthe donor moiety to the acceptor moiety results in changes in the energytransfer signal. The changes in the signal or the energy transfer signalfrom the donor or acceptor moiety can include changes in the: intensityof the signal; duration of the signal; wavelength of the signal;amplitude of the signal; polarization state of the signal; durationbetween the signals; or rate of the change in intensity, duration,wavelength or amplitude. The change in the signal or the energy transfersignal can include a change in the ratio of the change of the energytransfer donor signal relative to change of the energy transfer acceptorsignals. The signal or the energy transfer signal from the donor canincrease or decrease. In another example, the signal or the energytransfer signal from the acceptor can increase or decrease. The signalor the energy transfer signal associated with nucleotidetransient-binding can include: a decrease in the donor signal when thedonor is proximal to the acceptor; an increase in the acceptor signalwhen the acceptor is proximal to the donor; an increase in the donorsignal when the distance between the donor and acceptor increases; or adecrease in the acceptor signal when the distance between the donor andacceptor increases.

In an example, unincorporatable or non-incorporatable nucleotides oranalogs thereof may or may not have a structure similar to that of anative nucleotide which may include base, sugar, and phosphate moieties.

The non-incorporatable nucleotides can bind the polymerase/templatecomplex in a template-dependent manner, or can act as a universalmimetic and bind the polymerase/template complex in anon-template-dependent manner. The non-incorporatable nucleotides can bea nucleotide mimetic of incorporatable nucleotides, such as adenosine,guanosine, cytidine, thymidine or uridine nucleotides. Thenon-incorporatable nucleotide includes any compound having a nucleotidestructure, or a portion thereof, which can bind a polymerase.

For example, the non-incorporatable nucleotides can have the generalstructure:

R₁₁—(—P)_(n)—S—B

Where B can be a base moiety, such as a hetero cyclic base whichincludes substituted or unsubstituted nitrogen-containing heteroaromaticring. Where S can be a sugar moiety, such as a ribosyl, riboxyl, orglucosyl group. Where n can be 1-10, or more. Where P can be one or moresubstituted or unsubstituted phosphate or phosphonate groups. Where R11,if included, can be a reporter moiety (e.g., a fluorescent dye). In oneembodiment, the non-incorporatable nucleotide having multiple phosphateor phosphonate groups, the linkage between the phosphate or phosphonategroups can be non-hydrolyzable by the polymerase. The non-hydrolyzablelinkages include, but are not limited to, amino, alkyl, methyl, and thiogroups. Non-incorporatable nucleotide tetraphosphate analogs can havealpha-thio or alpha boreno substitutions.

The phosphate or phosphonate portion of the non-incorporatablenucleotide can have the general structure:

where B can be a base moiety and S can be a sugar moiety. Where any oneof the R1-R7 groups can render the nucleotide non-hydrolyzable by apolymerase. Where the sugar C5 position can be CH2, CH2O, CH═, CHR, orCH2. Where the R1 group can be O, S, CH═, CH(CN), or NH. Where the R2,R3, and R4, groups can independently be O, BH3, or SH. Where the R5 andR6 groups can independently be an amino, alkyl, methyl, thio group, orCHF, CF2, CHBr, CCl2, O—O, or —C≡C—. Where the R7 group can be oxygen,or one or more additional phosphate or phosphonate groups, or can be areporter moiety. Where R8 can be SH, BH3, CH3, NH2, or a phenyl group orphenyl ring. Where R9 can be SH. Where R10 can be CH3, N3CH2CH2, NH2,ANS, N3, MeO, SH, Ph, F, PhNH, PhO, or RS (where Ph can be a phenylgroup or phenyl ring, and F can be a fluorine atom or group). Thesubstituted groups can be in the S or R configuration.

The non-incorporatable nucleotides can be alpha-phosphate modifiednucleotides, alpha-beta nucleotide analogs, beta-phosphate modifiednucleotides, beta-gamma nucleotide analogs, gamma-phosphate modifiednucleotides, caged nucleotides, or di-nucleotide analogs.

In one aspect, nucleotides are compounds that can bind selectively to,or can be polymerized by, a polymerase. Typically, but not necessarily,the polymerase selectively binds the nucleotide and catalyzespolymerization of the nucleotide onto a nucleic acid strand (e.g.,nucleotide incorporation). Such nucleotides include not onlynaturally-occurring nucleotides but also any analogs, regardless oftheir structure, that can bind selectively to, or can be polymerized by,a polymerase. While naturally-occurring nucleotides typically comprisebase, sugar and phosphate moieties, the nucleotides of the presentdisclosure can include compounds lacking any one, some or all of suchmoieties.

The nucleotides can be operably linked to a reporter moiety (e.g.,labeled nucleotides) or can be un-labeled nucleotides. The nucleotidesalso include non-incorporatable nucleotides, and terminator nucleotides(e.g., chain terminating nucleotides and reversible terminatornucleotides). The nucleotides can be nucleotide polyphosphate molecules.Examples of nucleotide polyphosphate molecules and nucleosidepolyphosphate molecules include ribonucleotides, deoxyribonucleotides,ribonucleotide polyphosphate molecules, deoxyribonucleotidepolyphosphate molecules, peptide nucleotides, nucleoside polyphosphatemolecules, metallonucleosides, phosphonate nucleosides, and modifiedphosphate-sugar backbone nucleotides, and any analogs or variants of theforegoing.

The nucleotides typically comprise a chain of phosphorus atomscomprising three, four, five, six, seven, eight, nine, ten or morephosphorus atoms. In some embodiments, the phosphorus chain can beattached to any carbon of a sugar ring, such as the 2, 3, or 5′ carbon.The phosphorus chain can be linked to the sugar with an intervening O orS. One or more phosphorus atoms in the chain can be part of a phosphategroup having P and O. In another example, the phosphorus atoms in thechain can be linked together with intervening O, NH, S, methylene,substituted methylene, ethylene, substituted ethylene, CNH2, C(O),C(CH2), CH2CH2, or C(OH)CH2R (where R can be a 4-pyridine or1-imidazole). In one embodiment, the phosphorus atoms in the chain canhave side groups having O, BH3, or S. In the phosphorus chain, aphosphorus atom with a side group other than O can be a substitutedphosphate group. The phosphate groups include analogs, such asphosphoramidate, phosphorothioate, phosphorodithioate, andO-methylphosphoroamidite groups. At least one of the phosphate groupscan be substituted with a fluoro or chloro group. The phosphate groupscan be linked to the sugar moiety by an ester or phosphoramide linkage.

The nucleotides typically comprise a hetero cyclic base which includessubstituted or unsubstituted nitrogen-containing parent heteroaromaticring which is commonly found in nucleic acids, includingnaturally-occurring, substituted, modified, or engineered variants, oranalogs of the same. The base is capable of forming Watson-Crick orHoogstein hydrogen bonds with an appropriate complementary base.Exemplary bases include, but are not limited to, purines and pyrimidinessuch as: 2-aminopurine, 2,6-diaminopurine, adenine (A), ethenoadenine,N6-2-isopentenyladenine (6iA), N6-2-isopentenyl-2-methylthioadenine(2ms6iA), N6-methyladenine, guanine (G), isoguanine, N2-dimethylguanine(dmG), 7-methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine (6sG),hypoxanthine and O6-methylguanine; 7-deaza-purines such as7-deazaadenine (7-deaza-A) and 7-deazaguanine (7-deaza-G); pyrimidinessuch as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T),4-thiothymine (4sT), 5,6-dihydrothymine, O4-methylthymine, uracil (U),4-thiouracil (4sU) and 5,6-dihydrouracil (dihydrouracil; D); indolessuch as nitroindole and 4-methylindole; pyrroles such as nitropyrrole;nebularine; inosines; hydroxymethylcytosines; 5-methycytosines; base(Y); as well as methylated, glycosylated, and acylated base moieties;and the like.

The nucleotides typically comprise a suitable sugar moiety, such ascarbocyclic moiety, acyclic moieties, and other suitable sugar moieties.The sugar moiety may be selected from the following: ribosyl,2′-deoxyribosyl, 3′-deoxyribosyl, 2′,3′-dideoxyribosyl,2′,3′-didehydrodideoxyribosyl, 2′-alkoxyribosyl, 2′-azidoribosyl,2′-aminoribosyl, 2′-fluororibosyl, 2′-mercaptoriboxyl,2′-alkylthioribosyl, 3′-alkoxyribosyl, 3′-azidoribosyl, 3′-aminoribosyl,3′-fluororibosyl, 3′-mercaptoriboxyl, 3′-alkylthioribosyl carbocyclic,acyclic and other modified sugars.

Provided herein are labeled nucleotides which can bind in atemplate-dependent manner, to a nucleic acid-dependent polymerase. Inpracticing the methods provided herein, the incorporation of the labelednucleotides can be inhibited by any reaction condition which permitstransient binding of a nucleotide (e.g., complementary ornon-complementary) to a polymerase, and inhibits nucleotideincorporation, including: (1) reaction conditions and reagents (e.g.,temperature, pH, ionic strength, divalent cations, or time); (2)modified polymerases; (3) modifications of the nucleotide which inhibitincorporation; or (4) non-extendible polymerization initiation site.

The labeled nucleotide can bind the polymerase, which is bound to abase-paired template nucleic acid molecule and polymerization initiationsite. The polymerase can interrogate the labeled nucleotide forcomplementarity with the template nucleotide on the template molecule.The transient-binding time of the complementary labeled nucleotide canbe longer compared to the transient-binding time of thenon-complementary labeled nucleotide.

The labeled nucleotides comprise a nucleotide operably linked to atleast one reporter moiety at any position of the base or sugar, or anyof the phosphate groups (alpha, beta, gamma, any phosphate group distalto the sugar moiety, or a terminal phosphate group).

The labeled nucleotide can be a non-incorporatable nucleotide or aterminator nucleotide which is operably linked to at least one reportermoiety. The reporter moiety can be a fluorescent dye, energy transferdye, or any other type of reporter moiety. The labeled nucleotide can beoperably linked to different types of reporter moieties. The same typeor different types of reporter moieties can be operably linked todifferent types of nucleotides, for example, to permit base distinctionor identification. A linear or branched linker can be used to attach thenucleotide to the reporter moiety. An intervening linker can connectdifferent reporter moieties to each other or to the nucleotide in anycombination of linking arrangements. The labeled nucleotide can beincorporated by a naturally occurring, modified, or engineered nucleicacid-dependent polymerase. The labeled nucleotide can be resistant todegradation by 3′-5′ exonuclease activity of the polymerase.

Useful linking schemes include attaching reporter moieties tooligonucleotides synthesized using phosphoramidate to incorporateamino-modified dT. In one embodiment, the reporter moiety (e.g.,fluorophore) can be linked to the base of the nucleotide via ahexylacrylamide linker. In another embodiment, the reporter moiety(e.g., fluorophore) can be linked to the C5 position of the base (e.g.,cytosine or uracil) via a hexylacrylamide linker (Molecular Probes,A32763 and A32771), or can be linked to the C5 position of the base(e.g., uracil) via a propargylamino linker. In yet another embodiment,the reporter moiety (fluorophore) can be linked to the N7 position ofthe base (e.g., guanosine) via a propargylamino linker.

Provided herein are one or more reporter moieties which are operablylinked to the labeled nucleotides, terminator nucleotides,non-incorporatable nucleotides, nanoparticles, polymerases, templatenucleic acid molecules, primer molecules, surfaces, or oligonucleotides.

The reporter moiety generates, or causes to generate, a detectablesignal. Any suitable reporter moiety may be used, including luminescent,photoluminescent, electroluminescent, bioluminescent, chemiluminescent,fluorescent, phosphorescent, chromophore, radioisotope, electrochemical,mass spectrometry, Raman, hapten, affinity tag, atom, or an enzyme. Thereporter moiety generates a detectable signal resulting from a chemicalor physical change (e.g., heat, light, electrical, pH, saltconcentration, enzymatic activity, or proximity events). A proximityevent includes two reporter moieties approaching each other, orassociating with each other, or binding each other.

The reporter moieties may be selected so that each absorbs excitationradiation or emits fluorescence at a wavelength distinguishable from theother reporter moieties to permit monitoring the presence of differentreporter moieties in the same reaction. Two or more different reportermoieties can be selected having spectrally distinct emission profiles,or having minimal overlapping spectral emission profiles.

In one aspect, the signals from the different reporter moieties do notsignificantly overlap or interfere, by quenching, colorimetricinterference, or spectral interference.

The chromophore moiety may be 5-bromo-4-chloro-3-indolyl phosphate,3-indoxyl phosphate, p-nitrophenyl phosphate, and derivatives thereof,or lactamase or peroxidase based chemistry.

The chemiluminescent moiety may be a phosphatase-activated 1,2-dioxetanecompound. The 1,2-dioxetane compound includes disodium2-chloro-5-(4-methoxyspiro[1,2-dioxetane-3,2′-(5-chloro-)tricyclo[3,3,1-13,7]-decan]-1-yl)-1-phenylphosphate (e.g., CDP-STAR), chloroadamant-2′-ylidenemethoxyphenoxyphosphorylated dioxetane (e.g., CSPD), and3-(2′-spiroadamantane)-4-methoxy-4-(3″-phosphoryloxy)phenyl-1,2-dioxetane(e.g., AMPPD).

The fluorescent moiety includes: rhodols; resorufins; coumarins;xanthenes; acridines; fluoresceins; rhodamines; erythrins; cyanins;phthalaldehydes; naphthylamines; fluorescamines; benzoxadiazoles;stilbenes; pyrenes; indoles; borapolyazaindacenes; quinazolinones;eosin; erythrosin; Malachite green; CY dyes (GE Biosciences), includingCy3 (and its derivatives) and Cy5 (and its derivatives); DYOMICS andDYLIGHT dyes (Dyomics) including DY-547, DY-630, DY-631, DY-632, DY-633,DY-634, DY-635, DY-647, DY-649, DY-652, DY-678, DY-680, DY-682, DY-701,DY-734, DY-752, DY-777 and DY-782; Lucifer Yellow; CASCADE BLUE; TEXASRED; BODIPY (boron-dipyrromethene) (Molecular Probes) dyes includingBODIPY 630/650 and BODIPY 650/670; ATTO dyes (Atto-Tec) including ATTO390, ATTO 425, ATTO 465, ATTO 610 611X, ATTO 610 (N-succinimidyl ester),ATTO 635 (NHS ester); ALEXA FLUORS including ALEXA FLUOR 633, ALEXAFLUOR 647, ALEXA FLUOR 660, ALEXA FLUOR 700, ALEXA FLUOR 750, and ALEXAFLUOR 680 (Molecular Probes); DDAO(7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one or any derivativesthereof) (Molecular Probes); QUASAR dyes (Biosearch); IRDYES dyes(LiCor) including IRDYE 700DX (NHS ester), IRDYE 80016 (NHS ester) andIRDYE 800CW (NHS ester); EVOBLUE dyes (Evotech Biosystems); JODA 4 dyes(Applied Biosystems); HILYTE dyes (AnaSpec); MR121 and MR200 dyes(Roche); Hoechst dyes 33258 and 33242 (Invitrogen); FAIR OAKS RED(Molecular Devices); SUNNYVALE RED (Molecular Devices); LIGHT CYCLER RED(Roche); EPOCH (Glen Research) dyes including EPOCH REDMOND RED(phosphoramidate), EPOCH YAKIMA YELLOW (phosphoramidate), EPOCH GIGHARBOR GREEN (phosphoramidate); Tokyo green; and CF dyes including CF647 and CF555 (Biotium).

Quencher dyes may include: ATTO 540Q, ATTO 580Q, and ATTO 612Q(Atto-Tec); QSY dyes including QSY 7, QSY 9, QSY 21, and QSY 35(Molecular Probes); and EPOCH ECLIPSE QUENCHER (phosphoramidate) (GlenResearch). The fluorescent moiety can be a 7-hydroxycoumarin-hemicyaninehybrid molecule which is a far-red emitting dye.

The fluorescent moiety may be a fluorescence-emitting metal such as alanthanide complex, including those of Europium and Terbium.

Provided herein are reporter moieties which can be energy transfermoieties, such as energy transfer pairs (e.g., donors and acceptors),which are operably linked to the labeled nucleotides, terminatornucleotides, non-incorporatable nucleotides, nanoparticles, polymerases,template nucleic acid molecules, primer molecules, surfaces, oroligonucleotides.

In one aspect, the energy transfer moiety can be an energy transferdonor, such as a nanoparticle or an energy transfer dye. In anotheraspect, the energy transfer moiety can be an energy transfer acceptor,such as an energy acceptor dye.

In one aspect, the energy transfer pair can be operably linked to thesame molecule. In another aspect, the donor and acceptor can be operablylinked to different molecules in any combination. For example, the donorcan be linked to the polymerase, template molecule, or primer molecule,and the acceptor can be linked to the nucleotide (e.g.,non-incorporatable nucleotide or terminator nucleotide), the templatemolecule, or the primer molecule.

The energy transfer donor is capable of absorbing electromagnetic energy(e.g., light) at a first wavelength and emitting excitation energy inresponse. The energy acceptor is capable of absorbing excitation energyemitted by the donor and fluorescing at a second wavelength in response.

The donor and acceptor moieties can interact with each other physicallyor optically in a manner which produces a detectable signal when the twomoieties are in proximity with each other. A proximity event includestwo different moieties (e.g., energy transfer donor and acceptor)approaching each other, or associating with each other, or binding eachother.

The donor and acceptor moieties can transfer energy in various modes,including: fluorescence resonance energy transfer (FRET), scintillationproximity assays (SPA), luminescence resonance energy transfer (LRET),direct quenching, chemiluminescence energy transfer (CRET),bioluminescence resonance energy transfer (BRET), and excimer formation.

In one aspect, the energy transfer pair can be FRET donor and acceptormoieties. FRET is a distance-dependent radiationless transmission ofexcitation energy from a donor moiety to an acceptor moiety. Forexample, a donor moiety, in an excited state, transfers its energy to aproximal acceptor moiety by non-radiative dipole-dipole interaction orenergy transfer not strictly following the Forster's theory, such asnonoverlapping energy transfer occurring when nonoverlapping acceptorsare utilized. Typically, the efficiency of FRET energy transmission isdependent on the inverse sixth-power of the separation distance betweenthe donor and acceptor, which is approximately 10-100 Angstroms. FRET isuseful for investigating changes in proximity between or withinbiological molecules. FRET efficiency may depend on donor-acceptordistance r as 1/r6 or 1/r4. The distance where FRET efficiency is 50% istermed R0, also known as the Forster distance. R0 is unique for eachdonor-acceptor combination and may be about 5 to 10 nm. The efficiencyof FRET energy transfer can sometimes be dependent on energy transferfrom a point to a plane which varies by the fourth power of distanceseparation.

In biological applications, FRET can provide an on-off type signalindicating when the donor and acceptor moieties are within proximity ofeach other. Additional factors affecting FRET efficiency include thequantum yield of the donor, the extinction coefficient of the acceptor,and the degree of spectral overlap between the donor and acceptor.Procedures are well known for maximizing the FRET signal and detectionby selecting high yielding donors and high absorbing acceptors with thegreatest possible spectral overlap between the two. The change influorescence from a donor (e.g., reduced fluorescence signal) during aFRET event, can be an indication of proximity between a donor andacceptor moiety.

The production of signals from FRET donors and acceptors can besensitive to the distance between donor and acceptor moieties, theorientation of the donor and acceptor moieties, or a change in theenvironment of one of the moieties. For example, a nucleotide (e.g.,non-incorporatable or terminator nucleotide) linked with a FRET moiety(e.g., acceptor) may produce a detectable signal when it approaches,associates with, or binds a polymerase linked to a FRET moiety (e.g.,donor). In another example, a FRET donor and acceptor linked to oneprotein can emit a FRET signal upon conformational change of theprotein. Some FRET donor/acceptor pairs exhibit changes in absorbance oremission in response to changes in their environment, such as changes inpH, ionic strength, ionic type (NO2, Ca+2, Mg+2, Zn+2, Na+, Cl−, K+),oxygen saturation, and solvation polarity.

The FRET donor or acceptor may be a fluorophore, luminophore,chemiluminophore, bioluminophore, or quencher. Accordingly, the FRETdonor and acceptors may undergo fluorescence or other types of energytransfer with each other, including luminescence resonance energytransfer, bioluminescence resonance energy transfer, chemiluminescenceresonance energy transfer, and similar types of energy transfer notstrictly following the Forster's theory, such as the non-overlappingenergy transfer when non-overlapping acceptors are utilized.

The energy transfer moiety can be a FRET quencher. Typically, quenchershave an absorption spectrum with large extinction coefficients, howeverthe quantum yield for quenchers is reduced, such that the quencher emitslittle to no light upon excitation. Quenching can be used to reduce thebackground fluorescence, thereby enhancing the signal-to-noise ratio. Inone aspect, energy transferred from the donor may be absorbed by thequencher which emits moderated (e.g., reduced) fluorescence. In anotheraspect, the acceptor can be a non-fluorescent chromophore which absorbsthe energy transferred from the donor and emits heat (e.g., the energyacceptor is a dark quencher).

For an example, a quencher can be used as an energy acceptor with ananoparticle donor in a FRET system. One exemplary method involves theuse of quenchers in conjunction with reporters comprising fluorescentreporter moieties. In this strategy, certain nucleotides in the reactionmixture are labeled with a reporter comprising a fluorescent label,while the remaining nucleotides are labeled with one or more quenchers.Alternatively, each of the nucleotides in the reaction mixture islabeled with one or more quenchers. Discrimination of the nucleotidebases is based on the wavelength or intensity of light emitted from theFRET acceptor, as well as the intensity of light emitted from the FRETdonor. If no signal is detected from the FRET acceptor, a correspondingreduction in light emission from the FRET donor indicatestransient-binding of the nucleotide labeled with a quencher. The degreeof intensity reduction may be used to distinguish between differentquenchers.

Examples of fluorescent donors and non-fluorescent acceptor (e.g.,quencher) combinations have been developed for detection of proteolysisand nucleic acid hybridization. FRET donors, acceptors and quenchers canbe moieties that absorb electromagnetic energy (e.g., light) at about300-900 nm, or about 350-800 nm, or about 390-800 nm.

Energy transfer donor and acceptor moieties can be made from materialswhich typically fall into four general categories, including: (1)organic fluorescent dyes, dark quenchers and polymers (e.g.,dendrimers); (2) inorganic material such as metals, metal chelates andsemiconductors nanoparticles; (3) biomolecules such as proteins andamino acids (e.g., green fluorescent protein and derivatives thereof);and (4) enzymatically catalyzed bioluminescent molecules. The materialfor making the energy transfer donor and acceptor moieties can beselected from the same or different categories.

The FRET donor and acceptor moieties which are organic fluorescent dyes,quenchers or polymers include traditional dyes that emit in the UV,visible, or near-infrared region. The UV emitting dyes includecoumarin-, pyrene-, and naphthalene-related compounds. The visible andnear-infrared dyes include xanthene-, fluorescein-, rhodol-, rhodamine-,and cyanine-related compounds. The fluorescent dyes also includes DDAO((7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)), resorufin,ALEXA FLUOR and BODIPY dyes (both Molecular Probes), HILYTE Fluors(AnaSpec), ATTO dyes (Atto-Tec), DY dyes (Dyomics GmbH), TAMRA (PerkinElmer), tetramethylrhodamine (TMR), TEXAS RED, DYLIGHT (Thermo FisherScientific), FAM (AnaSpec), JOE and ROX (both Applied Biosystems), andTokyo Green.

Additional fluorescent dyes which can be used as quenchers includes:DNP, DABSYL, QSY (Molecular Probes), ATTO (Atto-Tec), BHQ (BiosearchTechnologies), QXL (AnaSpec), BBQ (Berry and Associates) and CY5Q/7Q(Amersham Biosciences).

The FRET donor and acceptor moieties which comprise inorganic materialsinclude gold (e.g., quencher), silver, copper, silicon, semiconductornanoparticles, and fluorescence-emitting metal such as a lanthanidecomplex, including those of Europium and Terbium.

Suitable FRET donor/acceptor pairs include: FAM as the donor and JOE,TAMRA, and ROX as the acceptor dyes. Other suitable pairs include: CYAas the donor and R6G, TAMRA, and ROX as the donor dyes.

In one embodiment, a nucleotide can be operably linked to a suitablereporter moiety. This labeled nucleotide can bind transiently to thepolymerase. The reporter moiety can be excited and the signal emitted bythe reporter moiety can be detected.

In another embodiment, a polymerase can be operably linked to a suitablenanoparticle and a nucleotide can be operably linked to a suitablereporter moiety. The labeled nucleotide can bind to the polymerase. Thenanoparticle can be excited and the resulting energy from the excitednanoparticle can be transferred to the reporter moiety. The transferredenergy can excite the reporter moiety, which can be emitted as adetectable signal.

Provided herein are terminator nucleotides which can be incorporatedonto the polymerization initiation site, in a template-dependent manner,by a nucleic acid-dependent polymerase, but the terminator nucleotide(which is incorporated) inhibits the incorporation of the nextnucleotide. The terminator nucleotide can be incorporated by a naturallyoccurring, modified, or engineered nucleic acid-dependent polymerase.

The extending strand comprises the polymerization initiation site and anucleotide which is incorporated at the terminal 3′ end of theinitiation site. The extending strand can have an extendible- ornon-extendible 3′ terminal end. The extendible end includes a terminal3′-OH group. The non-extendible end is not extendible by a polymerase.The non-extendible end can include any moiety which inhibitsincorporation of the next nucleotide. A terminator nucleotide, which isincorporated onto the polymerization initiation site, forms the newnon-extendible end of the extending strand.

The terminator nucleotide comprises a nucleotide operably linked to aninhibitor moiety. The inhibitor moiety comprises any chemical compoundor chemical group which permits incorporation of the terminatornucleotide by the polymerase but inhibits incorporation of the nextnucleotide. Thus, the polymerase can incorporate one and only oneterminator nucleotide, thereby advancing nucleotide incorporation byonly one base. The inhibitor moiety can be operably linked to anyportion of the nucleoside or nucleotide (e.g., any phosphate group, orbase or sugar moiety). The same type or different types of inhibitormoieties can be operably linked to different types of nucleotides. Theterminator nucleotide can be resistant to degradation by 3′-5′exonuclease activity of the polymerase.

On the terminator nucleotide, the inhibitor moiety can be modified sothat the next nucleotide can be incorporated (e.g., reversibleterminator nucleotide). Alternatively, the inhibitor moiety can beremoved (de-blocking) to permit incorporation of the next nucleotide.Accordingly, on a polymerization initiation site which has a terminatornucleotide incorporated onto its 3′ end, the inhibitor moiety can bemodified or removed to permit incorporation of the next nucleotide.

In one embodiment, the terminator nucleotides can be non-labeled, or canbe operably linked to at least one reporter moiety at any position ofthe base or sugar, or any of the phosphate groups (alpha, beta, gamma,or terminal phosphate group).

Suitable terminator nucleotides having inhibitor moieties attached tothe sugar 3′ position, base-linked dyes, where the linkers are cleavableunder the same conditions are useful. Suitable terminator nucleotideshaving photocleavable linkers are also useful.

The terminator nucleotides comprise a nucleotide operably linked to atleast one suitable inhibitor moiety. The inhibitor moiety comprises anychemical compound or chemical group which permits incorporation onto thepolymerization initiation site, in a template-dependent manner, by anucleic acid-dependent polymerase, but inhibits incorporation of thenext nucleotide. The inhibitor moiety can modify, substitute, orprotect, any portion of the nucleotide (e.g., base, sugar, or phosphategroup). A suitable inhibitor moiety can be operably linked to any partof the nucleotide (or nucleoside) including the base or sugar moiety, orany phosphate group. The suitable inhibitor moiety can permitincorporation of the terminator nucleotide, in a polymerase-driven,template-dependent manner, but inhibits, stalls, or slows downincorporation of the next nucleotide by the polymerase. The suitableinhibitor moiety inhibits incorporation of the next nucleotide byphysical, chemical, or charge interaction with the polymerase orincoming nucleotide.

The suitable inhibitor moiety can be operably linked to the 2′ or 3′position of the sugar moiety. In one embodiment, the 2′ or 3′-H or —OHgroup of the sugar moiety can be modified, substituted, or protected.For example, it is well known that DNA polymerases require apolymerization initiation site having a terminal 3′-OH group. Thus, theinhibitor moiety can be any chemical group or compound, which is not an—OH group, operably linked to the 3′ C of the sugar moiety. In someembodiments, the suitable inhibitor moiety can be an —H group operablylinked to the 3′ C of the sugar moiety. Such embodiments includedideoxynucleosides and dideoxynucleotides.

The suitable inhibitor moiety can be operably linked to any position ofthe nitrogenous base, such as a purine group, including the C2, C4, C5,N3, or C6, of cytosine, thymine, and uracil. The suitable inhibitormoiety can be operably linked to any position of the pyrimidine group,including the C2, C6, C8, N3 and N7 of adenine and guanine.

The suitable inhibitor moiety can be operably linked to any phosphategroup, such as the alpha, beta, gamma, or a terminal phosphate group.

In another embodiment, the suitable inhibitor moiety can be linked toany portion of the nucleoside or nucleotide, and sterically hinder theincoming nucleotide. In yet another embodiment, the suitable inhibitormoiety can be a charged group (positive or negative) and linked to anyportion of the nucleoside or nucleotide and can inhibit the polymerasefrom incorporating the next nucleotide. In another embodiment, thesuitable inhibitor moiety can be linked to at least one of: asterically-hindering group, fluorophore, or quencher, in any order andin any combination.

The suitable inhibitor moiety comprises any group including: amine,alkyl, alkenyl, alkynyl, alkyl amide, aryl, ether, ester, benzyl,propargyl, propynyl, phosphate, or analog thereof. For example, thesuitable inhibitor moiety can be a 3′-O-allyl moiety.

Suitable inhibitor moieties are well known in the art, and include:fluorenylmethyloxycarbonyl (FMOC), 4-(anisyl)diphenylmethyltrityl(MMTr), dimethoxytrityl (DMTr), monomethoxytrityl, trityl (Tr), benzoyl(Bz), isobutyryl (ib), pixyl (pi), ter-butyl-dimethylsilyl (TBMS), and1-(2-fluorophenyl)-4-methoxypiperidin 4-yl (FPMP).

The suitable inhibitor moiety can be a reporter moiety (e.g.,fluorescent dye) operably linked to the base or sugar moiety. Forexample, a fluorescent dye operably linked to the base via a2-nitrobenzyl group, where the 2-nitrobenyl group has the alpha carbonsubstituted with one alkyl or aryl group. The 2-nitrobenzyl group can bephotocleavable.

In another example, the suitable inhibitor moiety can be a reportermoiety (e.g., fluorescent dye, e.g., ALEXA FLUOR 549) operably linked tothe 5 position of pyrimidines or the 7 position of the purines, via acleavable disulfide linker.

In yet another example, the suitable inhibitor moiety can be arhodamine-type dyes, such as R6G, R110, ROX, or TAMRA, ordichloro-derivatives thereof, which are based-linked dyes, including thecommercially-available rhodamine dye terminator nucleotides from AppliedBiosystems. The suitable inhibitor moiety can be a charged group(positive or negative) or a group capable of becoming charged, includinga carboxylic acid, carboxylated, phosphate, di-phosphate, peptide,dipeptide, sulfate, disulfate, caproic acid, or amino acid (e.g., anegatively charged amino acid such as aspartic acid, glutamic acid,histidine, lysine, or arginine).

The suitable inhibitor moiety can be a non-incorporatable nucleotide ornucleoside which is linked to the base by a tether. The tether can belinked to a fluorescent label. The tether can include a cleavablemoiety, such as a disulfide group. The suitable inhibitor moiety can bea hydrocaryldithiomethyl-modified compound. The suitable inhibitormoiety can include an ethyl dithio linker. The suitable inhibitor moietycan be an alkyl chain homologue having any chain length, which can beproduced by replacing 2-bromoethanol and ethylsulfide reagents with anyalkyl chain homologue. The suitable inhibitor moiety can be anyphosphate, SO3, or C(O)R group, or modified groups thereof. In the C(O)Rgroup, R can be an H, alkyl, benzyl, aryl, alkenyl, alkynyl group, anycombination thereof.

In one embodiment, removal or modification of the inhibitor moiety whichis attached to the 3′ C of the sugar moiety, and restoration of a 3′-OHgroup, can permit incorporation of a subsequent nucleotide (e.g.,reversible terminator nucleotide). In another embodiment, removal ormodification of the inhibitor moiety which is attached to the sugar,base, or phosphate group, can permit incorporation of a subsequentnucleotide (e.g., reversible terminator nucleotide).

In one aspect, a suitable linker operably links the terminatornucleotide to the inhibitor moiety. The suitable linker does notinterfere with the function or activity of the nucleotide, nucleoside,or inhibitor moiety. The suitable linker can be cleavable orfragmentable to permit removal of the inhibitor moiety. The suitablelinker can be the inhibitor moiety. In one embodiment, the nucleotidecan be attached directly to the inhibitor moiety without an interveninglinker. Various linkers and linker chemistries for generating theterminator nucleotides are disclosed infra.

The terminator nucleotides can be linked to inhibitor moieties using anysuitable linking scheme, including linking schemes using amine linkers,or primary or secondary amines, or a rigid hydrocarbon arm.

The terminator nucleotide can include more than one linker, where thelinkers are the same or different. The multiple linkers can be removed,cleaved or fragmented using different temperatures, enzymaticactivities, chemical agents, or different wavelengths of electromagneticradiation.

In the terminator nucleotide, the suitable linker can be cleavable byheat, enzymatic activity, chemical agent, or electromagnetic radiation.Cleavable groups include: disulfide, amide, thioamide, ester, thioester,vicinal diol, or hemiacetal. Other cleavable bonds includeenzymatically-cleavable bonds, such as peptide bonds (cleaved bypeptidases), phosphate bonds (cleaved by phosphatases), nucleic acidbonds (cleaved by endonucleases), and sugar bonds (cleaved byglycosidases).

In one embodiment, the cleavable linker can be a photocleavable linker,such as a 2-nitrobenzyl linker, or others. Analogs of the 2-nitrobenzyllinker, and other photocleavable linkers can be used as cleavableblocking groups, including: 2-nitrobenzyloxycarbonyl (NBOC);nitroveratryl; 1-pyrenylmethyl; 6-nitroveratryloxycarbonyl (NVOC);dimethyldimethoxy-benzyloxycarbonyl (DDZ); 5-bromo-7-nitroindolinyl;O-hydroxy-alpha-methyl-cinnamoyl; methyl-6-nitroveratryloxycarbonyl;methyl-6-nitropiperonyloxycarbonyl; 2-oxymethylene anthraquinone;dimethoxybenzyloxy carbonyl; 5-bromo-7-nitroindolinyl;O-hydroxy-alpha-methyl cinnamoyl; t-butyl oxycarbonyl (TBOC), and2-oxymethylene anthriquinone. The photocleavable linkers can beilluminated with an electromagnetic source at about 320-800 nm,depending on the particular linker, to achieve cleavage. For example,1-(2-nitrophenyl)ethyl can be cleaved with light at about 300-350 nm,and 5-bromo-7-nitroindolinyl can be cleaved with light at about 420 nm.In another embodiment, the photocleavable linker can serve as theinhibitor moiety.

In another embodiment, the terminator nucleotide can include two or morecleavable linkers, each attached to a different portion of thenucleotide. For example, the terminator nucleotide can include twodifferent photo-cleavable linkers that are cleavable with the same ordifferent wavelengths of light.

In another embodiment, the linker can be an ethyl dithio or an alkylchain linker. In another embodiment, the cleavable linker can be adisulfide-linker which is a chemically-cleavable linker. In yet anotherembodiment, the cleavable linker can be an allyl moiety which iscleavable by palladium (Pd(0)) in a deallylation reaction, or anazidomethyl group which is cleavable with Tris(2-carboxyethyl)phosphine(TCEP) in aqueous solution. In still another embodiment, the linker canbe cleavable with silver nitrate (AgNO3). In another embodiment, anazidomethyl group can serve as an inhibitor moiety and a cleavablelinker.

A procedure for synthesizing a terminator nucleotide having an unblocked3′OH group and carrying a biotin molecule linked to the base moiety(N6-alkylated base) via a 2-nitrobenzyl linker can be envisaged.

In the terminator nucleotide, the suitable fragmentable linker iscapable of fragmenting in an electronic cascade self-eliminationreaction. In some embodiments, the fragmentable linker comprises atrigger moiety. The trigger moiety comprises a substrate that can becleaved or “activated” by a specified trigger agent. Activation of thetrigger moiety initiates a spontaneous rearrangement that results in thefragmentation of the linker and release of the enjoined compound. Forexample, the trigger moiety can initiate a ring closure mechanism orelimination reaction. Various elimination reactions, include 1,4-, 1,6-and 1,8-elimination reactions.

Any means of activating the trigger moiety may be used. Selection of aparticular means of activation, and hence the trigger moiety, maydepend, in part, on the particular fragmentation reaction desired. Insome embodiments, activation is based upon cleavage of the triggermoiety. The trigger moiety can include a cleavage site that is cleavableby a chemical reagent or enzyme. For example, the trigger moiety caninclude a cleavage recognition site that is cleavable by a sulfatase(e.g., SO3 and analogs thereof), esterase, phosphatase, nuclease,glycosidase, lipase, esterase, protease, or catalytic antibody.

In an example, photodiodes used for detection can be implemented suchthat they are fully depleted. Correlated double sampling is used toeliminate thermal noise of the pixel, creating a highly sensitive pixel.Read noise can be less than a few electrons. To increase sensitivityeven further, electron-multiplication (EM) gain can be used. Thestructure to enable EM gain can be implemented in the non-photosensitivearea of the pixel. A large electric field can be created betweenadjacent electrodes after charge is transferred from the photodiode orCCD. As the electric field becomes large enough, impact ionizationoccurs and the collected electrons are multiplied. This multiplicationprocess allows the pixel to have noise levels less than 1 electron suchthat the pixel is single photon sensitive. Furthermore, the pixels canbe arranged near the polymerase such that no optical occlusions occur inthe path of transduction, which can give a 100% fill factor, whichallows for high quantum efficiency.

FIG. 42 includes an illustration of exemplary method for sequencing. Forexample, a sequencing device is inserted into a system, as illustratedat 4202. The sequencing device can be placed in fluid communication witha fluidics system and in electrical communication with a computationalsystem. A target polynucleotide solution can be applied to thesequencing device, as illustrated at 4204. Optionally, the targetpolynucleotide solution can be applied prior to inserting the sequencingdevice into the system. In such a case, the target polynucleotide can belocalized within regions of a surface corresponding with pixels. Inanother example, enzymes may be localized to the surface and a targetpolynucleotide may be applied to the system and captured by the enzymeslocalized to the surface.

Once polynucleotide targets are localized within the system, solutionsincluding at least one type of labeled nucleotide can be contacted withat least one of the targets. For example, a mixed nucleotide solutionincluding at least two different nucleotide types, each type modifiedwith a different dye having a different fluorescent spectrum, can flowthrough the flow cell of the sequencing device, as illustrated 4206. Ina particular example, a nucleotide solution including four nucleotidetypes, each type modified with a different associated dye havingdifferent emission spectrum, can be applied to the sequencing device. Asa result of incorporation of nucleotides along a target polynucleotide,fluorescence can be detected from the fluorescent dye associated withthe types of nucleotides, as illustrated at 4208. For example, thesolution including at least two dye modified nucleotide types isapplied, incorporation of the two different types can be detected basedon fluorescence at two different spectrums.

As illustrated 4210, the sequence of nucleotide incorporationsassociated with fluorescent emissions can be used to determine the baseidentities of one or more incorporated nucleotides. For example, a basecalling subroutine can determine, based on the sequence of detectedfluorescent signals detected by a single pixel, a sequence of nucleotidebases along a target polynucleotide.

In another exemplary method illustrated in FIG. 43, a sequencing devicecan be inserted into the system, as illustrated at 4302. A targetpolynucleotide solution can be applied to a sequencing device, asillustrated at 4304. As above, the target polynucleotide can be appliedprior to inserting the sequencing device in the system when the targetpolynucleotide is to be tethered to a surface. Alternatively, an enzymetethered to the surface of the polynucleotide is provided prior toflowing nucleotide solutions and the sequencing device.

The nucleotide solutions each including a different type of dye-modifiednucleotide can flow sequentially through the sequencing device, asillustrated at 4306. Optionally, four different nucleotide solutionseach including a different type of nucleotide modified with a dye can beutilized sequentially, flowing a different nucleotide solution betweenflows of a wash solution. Fluorescent emissions emitted as a result ofnucleotide incorporation can be detected, as illustrated 4308. In aparticular example in which four different nucleotide solutions areutilized, a fluorescent emission can indicate incorporated base based onthe solution flowing at the time the emitted fluorescence. As such, asingle dye can be used. Alternatively, multiple dyes can be used.

As a result of the detection, a nucleotide sequence can be determined,as illustrated at 4310. The sequence of fluorescent signals associatedwith the nucleotide solution flowing at time of a fluorescent signal canbe utilized to determine the sequence of nucleotides incorporated duringtemplate-dependent synthesis driven by a given target polynucleotidethat is associated with a single pixel.

Advantageously, a system can be formed without an external microscopeobjective and EM-CCDs and instead with a CMOS photosensitive substrate.In particular, the system advantageously includes a high density arrayof incorporation detectors integrated on a CMOS substrate. Optionally,the system includes immobilization of the enzyme, such as a DNApolymerase, at each detector or immobilization of the DNA strand at eachdetector. Each incorporation detector can contains an integratedradiation source as well as the spectrally sensitive detector. Spectralselectivity can be obtained by multiple confinement wells placed atvarious depths within the same pixel, can be implemented with permanentuse color filter photoresist, or can be implemented with absorptionlayers tuned with thickness. The excitation light source can beintegrated local to each incorporation site and bound within thesemiconductor substrate using hot carrier direct recombination. Data foreach incorporation site may be limited to 3 bits indicating anincorporation event along with the color.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, apparatuses and devices, fordetecting a signal, or a change in a signal, emitted by a labelednucleotide in a nucleotide incorporation reaction.

In some embodiments, a method for sequencing a target polynucleotide,comprises the steps of: contacting a device with an enzymatic reaction,wherein the enzymatic reaction includes contacting: (i) a polymerasewith (ii) at least one target polynucleotide which is base paired with aprimer and with (iii) at least one type of a nucleotide having anoptically detectable moiety, thereby incorporating a nucleotide onto theprimer, wherein the device comprises any of the integrated devicesdescribed herein that have detectors that detect signals from anucleotide incorporation reaction. The method further comprises thesteps of: generating an optically detectable signal by exciting theoptically detectable moiety with an excitation source; and detecting theoptically detectable signal. The method further comprises the steps of:identifying the incorporated nucleotide.

In some embodiments, a method for sequencing a target polynucleotidefurther comprises a second enzymatic reaction including the steps of:contacting the device with a second enzymatic reaction, wherein thesecond enzymatic reaction includes contacting: (i) a second polymerasewith (ii) the at least one target polynucleotide which is base pairedwith the primer and with (iii) at least one type of a nucleotide havingan optically detectable moiety, thereby incorporating a secondnucleotide onto the primer. The method can further comprise the stepsof: generating a second optically detectable signal by exciting thesecond optically detectable moiety with an excitation source; anddetecting the second optically detectable signal. The method furthercomprises the steps of: identifying the incorporated nucleotide.

Alternatively, the method for sequencing further comprises a differentsecond enzymatic reaction, comprising: contacting the device with asecond enzymatic reaction, wherein the second enzymatic reactionincludes contacting: (i) a second polymerase with (ii) the at least onetarget polynucleotide which is base paired with the primer and with(iii) at least one type of a nucleotide lacking an optically detectablemoiety, thereby incorporating onto the primer the at least one type ofnucleotide lacking an optically detectable moiety. This embodiment isknown as “dark” sequencing.

In some embodiments, a method for sequencing a target polynucleotidefurther comprises a third enzymatic reaction including the steps of:contacting the device with a third enzymatic reaction, which includes atleast one type of a second nucleotide lacking an optically detectablemoiety; and incorporating onto the primer the second nucleotide lackingthe optically detectable moiety.

Alternatively, the method for sequencing further comprises a differentthird enzymatic reaction, comprising: contacting the device with a thirdenzymatic reaction, wherein the enzymatic reaction includes at least onetype of a nucleotide having an optically detectable moiety; andgenerating a second optically detectable signal by exciting theoptically detectable moiety with an excitation source; detecting thesecond optically detectable signal; and identifying the secondincorporated nucleotide.

In some embodiments, a method for sequencing a target polynucleotidefurther comprises a fourth enzymatic reaction including the steps of:contacting the device with a fourth enzymatic reaction, wherein theenzymatic reaction includes at least one type of a nucleotide having anoptically detectable moiety; generating a second optically detectablesignal by exciting the optically detectable moiety with an excitationsource; detecting the second optically detectable signal; andidentifying the second incorporated nucleotide.

In some embodiments, any combination of the first, second, third andfourth nucleic sequencing reactions can be conducted with (i) at leastone type of nucleotide linked to an optically-detectable moiety such asan energy transfer acceptor moiety, and (ii) a polymerase linked to anenergy transfer donor moiety. In some embodiments, any combination ofthe first, second, third and fourth nucleic sequencing reactions can beconducted with a mutant polymerase having altered nucleotideincorporation kinetics, which include: altered polymerase binding to thetarget molecule, altered polymerase binding to the nucleotide, alteredpolymerase catalyzing nucleotide incorporation, altered the polymerasecleaving the phosphate group or substituted phosphate group, or alteredpolymerase releasing the cleavage product.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, apparatuses and devices, forgenerating an energy transfer signal comprising the steps of: contactinga device with an enzymatic reaction, where the enzymatic reactionincludes contacting: (i) a polymerase linked to an energy transfer donormoiety with (ii) a target nucleic acid molecule which is base-pairedwith a primer and with (iii) at least one type of a nucleotide having anenergy transfer acceptor moiety, thereby incorporating the nucleotideonto the primer, and locating the polymerase and the at least one typeof nucleotide in close proximity with each other to generate an energytransfer signal, wherein the device comprises any of the integrateddevices described herein that have detectors that detect signals from anucleotide incorporation reaction.

In some embodiments, for energy transfer sequencing methods, thepolymerase is a mutant polymerase having altered nucleotideincorporation kinetics. In some embodiments, the mutant polymerase hasaltered nucleotide incorporation kinetics, which include: alteredpolymerase binding to the target molecule, altered polymerase binding tothe nucleotide, altered polymerase catalyzing nucleotide incorporation,altered the polymerase cleaving the phosphate group or substitutedphosphate group, or altered polymerase releasing the cleavage product(see e.g., U.S. published application No. 2012/0322057, published Dec.20, 2012, hereby incorporated by reference in its entirety). In someembodiments, the energy transfer donor moiety comprises a nanoparticle(U.S. Pat. No. 8,603,792, issued Dec. 10, 2013, to Nikiforov, herebyincorporated by reference in its entirety). In some embodiment, thesequencing reaction is conducted with a single target nucleic acidmolecule (target polynucleotide). In some embodiments, the mutantpolymerase comprises the amino acid sequence found in any of SEQ IDNOS:1-3 of U.S. published application No. 2012/0329042, published Dec.27, 2012 (hereby incorporated by reference in its entirety).

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, apparatuses and devices, fordetecting a signal, or a change in a signal, emitted by a labelednucleotide which is transiently-bound to a polymerase and the templatenucleotide. The detected signal can be used to identify thetransiently-bound nucleotide and deduce the identity of the boundnucleotide.

The methods can be practiced using any suitable reaction conditioncomprising reagents and components which mediate polymerase-dependentreactions, including: forming the complex (template molecule/initiationsite/polymerase); transient-binding a labeled nucleotide to a polymerasein a template-dependent manner (without nucleotide incorporation); anddetecting the signal (or change in a signal) from the transiently-boundlabeled nucleotide. The methods can include a separate step forincorporating a nucleotide. In some embodiments, transiently-binding anucleotide to a polymerase, without polymerizing, includes not forming acovalent bond between the transiently bound nucleotide and a free 3?-OHend of a primer or nucleic acid molecule. The methods can be practicedusing one or more different types of polymerases. For example, themethods can be practiced using one type of polymerase for thetransient-binding step, and the same or different type of polymerase forthe nucleotide incorporation step. The methods can be practiced usingone or more different types of nucleotides. The methods can be practicedusing separate, step-wise reactions: contacting, binding, detecting,incorporating, or removing.

The transient-binding methods can be performed on any type ofpolymerase-dependent platform, including: single molecule, arrays ofsingle molecules, populations of immobilized template molecules (i.e.,multiple copies of the same template molecule immobilized on a device,support, solid surface or bead, etc), direct excitation/detection,FRET-based excitation/detection, fluorescence polarization,non-immobilized polymerase/template complex, or any combination thereof(see U.S. Pat. No. 8,632,975, issued on Jan. 21, 2014 to Vander Horn,hereby incorporated by reference in its entirety).

In some embodiments, methods for transiently binding a nucleotide to apolymerase can be conducted under any reaction condition which permitsthe polymerase to selectively bind a complementary nucleotide, butincorporation of the complementary nucleotide is perturbed, impeded, orinhibited. Such reaction conditions include utilizing: (1) any reactionconditions and reagents, such as temperature, pH, ionic strength,multivalent cations, or time; (2) any polymerase which selectively bindsa complementary nucleotide but exhibits reduced nucleotide incorporationactivity; (3) non-incorporatable nucleotides; or (4) a non-extendiblepolymerization initiation site. Any combination of these reactionconditions can be practiced in any order in the transient-bindingmethods provided herein.

In some embodiments, methods for detecting the presence of atransiently-bound nucleotide comprise the steps of: (a) contacting adevice with an enzymatic reaction, wherein the enzymatic reactioncomprises: contacting at least one type of a labeled nucleotide to acomplex having a first polymerase bound to at least one targetpolynucleotide that is bound to a primer, under suitable conditions totransiently-bind, without polymerizing, the at least one type of labelednucleotide to the polymerase in a nucleic acid template-dependentmanner; (b) detecting the transiently-bound labeled nucleotide; and (c)identifying the labeled nucleotide transiently-bound to the polymerase,wherein the device comprises any of the integrated devices describedherein that have detectors that detect signals from a nucleotideincorporation reaction. The steps (a)-(c) can be repeated at least once.In some embodiments, the method further comprises the steps of: (d)removing the transiently-bound nucleotide; and (e) contacting thecomplex with at least one type of a second nucleotide under suitableconditions for a second polymerase to polymerize the second nucleotide.The steps (d)-(e) can be repeated at least once. The steps (a)-(e) canbe repeated at least once.

In some embodiments, the suitable conditions for transiently-binding anucleotide include conducting the enzymatic reaction in the presence ofa cation that inhibits nucleotide incorporation by the polymerase. Insome embodiments, the labeled nucleotide comprises a nucleotide linkedto an optically detectable moiety. In some embodiments, the methodsinclude: generating an optically detectable signal by exciting theoptically detectable moiety with an excitation source; and detecting theoptically detectable signal. In some embodiments, the methods furthercomprise: identifying the incorporated nucleotide (the secondnucleotide). In some embodiments, the incorporated nucleotide produces anucleotide incorporation byproduct. The nucleotide incorporationbyproduct includes pyrophosphate, hydrogen ions or protons. In someembodiments, the methods further comprise: detecting the nucleotideincorporation byproduct. In some embodiment, the sequencing reaction isconducted with a single target nucleic acid molecule (targetpolynucleotide).

In some embodiments, methods for detecting the presence of atransiently-bound nucleotide comprise the steps of: (a) contacting adevice with an enzymatic reaction, wherein the enzymatic reactioncomprises: contacting at least one type of a labeled nucleotide to acomplex which comprises a polymerase bound to a template nucleic acidmolecule which is base-paired to a primer having a polymerizationinitiation site, under conditions suitable to transiently-bind thelabeled nucleotide to the polymerase in a template-dependent manner butinhibits incorporation of the nucleotide; (b) exciting the labelednucleotide or the polymerase with an excitation source; and (c)detecting a signal, or a change in a signal, emitted by thetransiently-bound labeled nucleotide in step (a), thereby detecting thepresence of the transiently-bound nucleotide, wherein the devicecomprises any of the integrated devices described herein that havedetectors that detect signals from a nucleotide incorporation reaction.In some embodiments, methods further comprise the step: (d) identifyingthe nucleotide transiently-bound to the polymerase. The steps (a)-(c)can be repeated at least once. The steps (a)-(d) can be repeated atleast once.

In one embodiment, the methods for identifying a nucleotide bound to apolymerase, further comprises the steps of: (e1) removing thetransiently-bound nucleotide; and (f1) contacting the complex with atleast one type of nucleotide under suitable conditions for thepolymerase to polymerize the nucleotide. In this embodiment, the samepolymerase in step (a) is contacted in step (f1). The steps (e1)-(f1)can be repeated at least once. The steps (a)-(f1) can be repeated atleast once. In some embodiments, the incorporated nucleotide produces anucleotide incorporation byproduct. The nucleotide incorporationbyproduct includes pyrophosphate, hydrogen ions or protons. In someembodiments, the methods further comprise: detecting the nucleotideincorporation byproduct.

In another embodiment, the methods for identifying a nucleotide bound toa polymerase, further comprises the steps of: (e2) removing the firstpolymerase and the transiently-bound nucleotide so that the templatenucleic acid molecule, nucleic acid primer molecule or self-primingtemplate nucleic acid molecule remains immobilized to the surface; (f2)binding the remaining template nucleic acid molecule with a secondpolymerase; and (g2) contacting the second polymerase with at least onetype of nucleotide under suitable conditions for the second polymeraseto polymerize the nucleotide. In this embodiment, the polymerase in step(a) is different from the polymerase in step (f2). The steps (e2)-(g2)can be repeated at least once. The steps (a)-(g2) can be repeated atleast once. In some embodiments, the incorporated nucleotide produces anucleotide incorporation byproduct. The nucleotide incorporationbyproduct includes pyrophosphate, hydrogen ions or protons. In someembodiments, the methods further comprise: detecting the nucleotideincorporation byproduct.

In one embodiment, the suitable conditions to transiently bind thenucleotide to the polymerase in step (a1) or (a2) comprise: (i) reducingthe levels or omission of a cation that permits nucleotide incorporationor addition of a cation that inhibits nucleotide incorporation; (ii) thepolymerase selectively binds the nucleotide in a template-dependentmanner and exhibits reduced nucleotide incorporation activity; (iii) theat least one type of labeled nucleotide is a labeled non-incorporatablenucleotide; or (iv) the polymerization initiation site is anon-extendible polymerization initiation site. Any combination of thesesuitable conditions can be practiced to identify the nucleotide bound tothe polymerase.

In another embodiment, the suitable conditions in step (a) comprise: (i)cations present at a concentration that inhibits nucleotideincorporation; (ii) the polymerase selectively binds the nucleotide in atemplate-dependent manner and exhibits reduced nucleotide incorporationactivity; (iii) the at least one type of labeled nucleotide is a labelednon-incorporatable nucleotide; or (iv) the polymerization initiationsite is a non-extendible polymerization initiation site.

In one embodiment, the cation that inhibits nucleotide incorporation canbe calcium, scandium, titanium, vanadium, chromium, iron, cobalt,nickel, copper, zinc, gallium, germanium, arsenic, selenium, rhodium, orstrontium.

In another embodiment, the suitable conditions for polymerizing thenucleotide in step (f1) or (g2) comprise: (i) including a cation thatpermits nucleotide incorporation or reducing the levels or omission of acation that inhibits nucleotide incorporation; (ii) using a polymerasewhich selectively binds the nucleotide in a template-dependent mannerand polymerizes the bound nucleotide; (iii) using at least one type ofincorporatable nucleotide; or (iv) using a polymerization initiationsite having an extendible polymerization initiation site. In someembodiments, cations that permit nucleotide incorporation includemagnesium and manganese.

In one embodiment, suitable conditions for incorporating the terminatornucleotide in any of steps (a) and (k) can include a manganese ormagnesium compound, or the manganese or magnesium compound can beomitted. In another embodiment, the manganese compound can be MnCl2. Inanother embodiment, the magnesium compound can be MgCl2. In anotherembodiment, the amount of manganese or magnesium compound can be about1-5 mM, or about 2-5 mM. In another embodiment, the manganese ormagnesium compound can be washed away or chelated after any of thesteps.

In some embodiments, the polymerase can be an RB69, Phi29, B103polymerase, or a Klenow fragment. In some embodiments, the polymerasecan exhibit exonuclease activity. In another embodiment, the polymerasecan be any 9° N polymerase or derivative thereof, including THERMINATOR,THERMINATOR II, or THERMINATOR-GAMMA polymerase (New England Biolabs,catalog # s M0261L, M0266L, and M0334L, respectively). In anotherembodiment, the second polymerase can be the same type or a differenttype as the first polymerase. In some embodiments, the polymerasecomprise a mutant polymerase, that can have altered nucleotideincorporation kinetics. In some embodiments, the altered nucleotideincorporation kinetics include: altered polymerase binding to the targetmolecule, altered polymerase binding to the nucleotide, alteredpolymerase catalyzing nucleotide incorporation, altered the polymerasecleaving the phosphate group or substituted phosphate group, or alteredpolymerase releasing the cleavage product. In some embodiments, thepolymerase comprises the amino acid sequence found in any of SEQ ID NOS:1-8 found in U.S. published application No. 2010/031114, published Dec.9, 2010 (hereby incorporated by reference in its entirety).

In some embodiments, any of the nucleic acid sequencing methodsdescribed herein include an enzyme that catalyzes polymerization of anucleotide in a template-dependent manner, including a DNA-dependentpolymerase, RNA-dependent polymerase, or a reverse transcriptase. Insome embodiments, the polymerase can be a wild-type (e.g., native) ormodified/mutant polymerase. In some embodiments, the polymerase can be athermolabile or thermostable polymerase. In some embodiments, thepolymerase lacks exonuclease activity. In some embodiments, thepolymerase can bind a labeled nucleotide. In some embodiments, thepolymerase can bind an incorporatable or a non-incorporatablenucleotide. In some embodiments, the first or the second polymerase canbe operably linked to a reporter moiety (e.g., energy transfer donormoiety).

In some embodiments, any of the nucleic acid sequencing methodsdescribed herein include an energy transfer donor moiety, such as afluorescent dye or nanoparticle (U.S. Pat. No. 8,603,792, issued Dec.10, 2013, to Nikiforov, hereby incorporated by reference in itsentirety). In some embodiments, the nanoparticle comprises an inorganicfluorescent nanoparticle. In some embodiments, the nanoparticle is 1-20nm in its largest dimension. In some embodiments, the nanoparticle is anon-blinking nanoparticle.

In some embodiments, any of the nucleic acid sequencing methodsdescribed herein include a template polynucleotide, which includes a DNAmolecule, RNA molecule, or DNA/RNA hybrid molecule. The templatepolynucleotide comprises a single template nucleic acid molecule or aplurality of template nucleic acid molecules. The plurality of templatenucleic acid molecules (target polynucleotides) comprises a populationof nucleic acids having different sequences or having substantiallyidentical sequences.

In some embodiments, any of the nucleic acid sequencing methodsdescribed herein include a primer having a polymerization initiationsite at the 3′ terminal end. The primer can be hybridized to thetemplate polynucleotide. The primer can be a self-priming templatenucleic acid molecule. In another embodiment, the polymerizationinitiation site can be base-paired to the template nucleic acidmolecule. In another embodiment, the polymerization initiation site canbe an extendible terminal 3′OH group or a non-extendible terminal group.In another embodiment, the polymerization initiation site can be aterminal 3′OH group of the nucleic acid primer molecule or a terminal3′OH group of a self-priming template nucleic acid molecule.

In some embodiments, any of the nucleic acid sequencing methodsdescribed herein include one or more nucleic acid molecules attached(immobilized) to a device, support, surface or microsphere. For example,a template polynucleotide, nucleic acid primer molecule, or self-primingtemplate polynucleotide, can be immobilized to a device, support,surface or microsphere. In some embodiments, the device is anyintegrated device described herein, including those having detectorsthat detect signals from a nucleotide incorporation reaction. In someembodiments, the support includes a particle or microsphere.

In some embodiments, any of the nucleic acid sequencing methodsdescribed herein include at least one type of nucleotide. The nucleotidecan be adenosine, guanosine, cytosine, thymidine, uridine or inosine. Insome embodiments, the nucleotide includes 3-10 phosphate groups, ormore.

In some embodiments, any of the nucleic acid sequencing methodsdescribed herein can be conducted with one type of nucleotide, or nomore than two different types of nucleotides, or no more than threedifferent types of nucleotides, or no more than four different types ofnucleotides, or more than four different types of nucleotides.

In some embodiments, the nucleotide can be non-labeled, or can be linkedto at least one reporter moiety. The reporter moiety includes anyoptically detectable moiety, such as a fluorophore, energy transferdonor moiety or energy transfer acceptor moiety. In some embodiments,the optically detectable moiety is photobleachable. In some embodiments,the optically-detectable moiety is attached to any portion of thenucleotide, including the base, sugar (e.g., 2′ or 3′ position) orphosphate backbone. The optically detectable moiety is attached to thenucleotide by a linker. In some embodiments, the linker is cleavablewith light, heat, a chemical compound or an enzyme. In some embodiments,the cleavable linker can be cleaved with light, heat, a chemicalcompound or an enzyme.

In some embodiments, the energy transfer donor reporter moiety can be aninorganic nanoparticle or a fluorophore. The energy transfer acceptormoiety can be a fluorophore (e.g., fluorescent dye). The energy transferdonor reporter moiety can be linked to the polymerase or nucleotide. Theenergy transfer acceptor reporter moiety can be linked to the polymeraseor nucleotide. In conducting any of the sequencing methods describedherein, as an example, a donor-labeled polymerase binds anacceptor-labeled nucleotide, thereby bringing the polymerase andnucleotide in close proximity to permit energy transfer from the donorto the acceptor and generation of an energy transfer signal. Uponexcitation from an excitation source (e.g., electromagnetic energy), theacceptor moiety emits a fluorescent signal.

The nucleotide can be incorporatable or non-incorporatable by apolymerase. The non-incorporatable nucleotide can bind to the polymeraseand template nucleic acid molecule which can be base-paired to apolymerization initiation site, in a template-dependent manner, but doesnot incorporate.

In some embodiments, any of the nucleic acid sequencing methodsdescribed herein include at least one nucleotide linked to at least oneinhibitor moiety to generate a terminator nucleotide. The inhibitormoiety (e.g., blocking moiety) can permit incorporation of theterminator nucleotide but inhibits incorporation of a subsequentnucleotide. The inhibitor moiety can be linked to any portion of thenucleotide including the base, sugar (e.g., 2′ or 3′ position) orphosphate backbone. The inhibitor moiety can be linked to the nucleotideby a cleavable linker that is cleavable with an enzyme, heat, chemicalcompound, or light. In another embodiment, the inhibitor moiety can beremoved via an enzymatic, heat, chemical or light cleavage reaction.

In some embodiments, any of the nucleic acid sequencing methodsdescribed herein include at least one nucleotide that is adideoxyribonucleotide.

In some embodiments, any of the nucleic acid sequencing methodsdescribed herein can be conducted by contacting a polymerase with amixture of nucleotides. The mixture can include different types ofnucleotides that differ from each other in their base, sugar orphosphate backbone. The mixture can include only labeled nucleotides, oronly non-labeled nucleotides, or at least one labeled (e.g., opticallydetectable moiety) and at least one non-labeled nucleotide. The mixturecan include two, three, four, or more different types of nucleotides.For example, the mixture can include: (i) one type of nucleotide havingan optically detectable moiety and two or three different types ofnucleotides that lack an optically detectable moiety; or (ii) twodifferent types of nucleotides having different optically detectablemoieties and one or two different types of nucleotides that lack anoptically detectable moiety; or (iii) three different types ofnucleotides having different optically detectable moieties and one typeof nucleotide that lacks an optically detectable moiety. In conducting anucleic acid sequencing reaction, the polymerase can be contacted withthe different types of nucleotide in the mixture of nucleotidesessentially simultaneously or sequentially.

In some embodiments, any of the nucleic acid sequencing methodsdescribed herein include an excitation source for exciting theoptically-detectable moiety. The excitation source includeselectromagnetic energy or light.

In some embodiments, any of the nucleic acid sequencing methodsdescribed herein can be conducted on a single site on a device, or at aplurality of sites on the device. The plurality of sites on the devicecan be arranged in an organized or random array. The plurality of siteson the device can be arranged in rectilinear or hexagonal pattern.

In some embodiments, any of the nucleic acid sequencing methodsdescribed herein can be conducted in a massively parallel manner. Thenucleic acid sequencing reactions can be conducted by subjecting aplurality of target polynucleotides to the same enzymatic reaction inparallel, wherein the plurality of target polynucleotides comprises apopulation of nucleic acids having substantially identical sequences ordifferent sequences.

In some embodiments, any of the nucleic acid sequencing methodsdescribed herein include nucleotide incorporation reactions whichproduce one or more nucleotide incorporation byproducts resulting fromthe polymerase catalyzing nucleotide polymerization. In someembodiments, nucleotide incorporation byproducts include pyrophosphatemolecules, hydrogen ions, or protons.

In some embodiments, any of the nucleic acid sequencing methodsdescribed herein can be conducted by flowing reagents to the surface ofthe integrated device, where the flow contains at least one reagent forconducting nucleic acid sequencing reactions, including polymerases, onetype of nucleotides or a mixture of different types of nucleotides,cations that inhibit nucleotide incorporation, cations that permitnucleotide incorporation, and cleaving agents. The flow can deliver tothe surface of the device multiple different reagents sequentially oressentially simultaneously.

Each of the above sequencing techniques can be used in conjunction withembodiments of the above described devices and systems.

In a first aspect, a device includes a transparent layer defining asurface exposed to a flow volume and to secure a target polynucleotidetemplate and a detector structure in optical communication with andsecured to the transparent layer and including a plurality of detectorsconfigured to detect a fluorescent signal emitted during nucleotideincorporation during template-dependent nucleic acid synthesis.

In an example of the first aspect, the detector structure includes aplurality of pixels, each pixel of the plurality of pixels including atleast two detectors of the plurality of detectors. For example, the atleast two detectors are disposed adjacent one anther within a plan view.In another example, the at least two detectors are disposed one over theother when viewed in cross-section. In a further example, each pixelincludes at least three detectors. For example, each pixel can includeat least four detectors.

In another example of the first aspect and the above examples, thetransparent layer includes an energy propagation layer.

In a further example of the first aspect and the above examples, thedevice further includes an energy propagation layer disposed between thetransparent layer and the detector structure. For example, the energypropagation layer includes a total internal reflection layer. In anotherexample, the device further includes an energy emitting component toprovide energy to the energy propagation layer.

In an additional example of the first aspect and the above examples, thedevice further includes a separator structure extending from thedetector structure toward the transparent layer, the separator structureopaque to the fluorescent signal.

In another example of the first aspect and the above examples, thedevice further includes a filter layer disposed between the devicestructure and the transparent layer. For example, the filter layer isconfigured to limit transmission of excitation energy. In anotherexample, the filter layer is configured to permit the transmission of awavelength spectrum associated with a dye.

In a further example of the first aspect and the above examples, thedevice further includes a well structure defining wells disposed on thetransparent layer opposite the detector structure.

In an additional example of the first aspect and the above examples, thedevice further includes a pad structure disposed on the transparentlayer opposite the detector structure.

In another example of the first aspect and the above examples, thedevice further includes a lid, the flow volume defined between the lidand the transparent layer.

In a further example of the first aspect and the above examples, thetransparent layer comprises an electrode.

In a second aspect, an apparatus includes a transparent layer defining asurface exposed to a flow volume and to secure a target polynucleotidetemplate; an energy propagation layer disposed opposite the surface ofthe transparent layer to propagate photonic energy along a path parallelto the surface; an excitation filter layer secured to the energypropagation layer opposite the transparent layer, the excitation filterlayer opaque to the photonic energy; and a detector structure secured tothe excitation filter layer opposite the energy propagation layer, thedetector structure defining a plurality of pixels, each pixel includinga detector, each pixel of the plurality of pixels uniquely opticallyassociated with a well of the plurality of wells.

In an example of the second aspect, each pixel includes at least twodetectors. For example, the at least two detectors can be disposedadjacent one another within a plan view. In another example, the atleast two detectors are disposed one over the other when viewed incross-section. In an additional example, each pixel includes at leastthree detectors. For example, each pixel can include at least fourdetectors. In a further example, the energy propagation layer includes atotal internal reflection layer. For example, the apparatus can furtherinclude an energy emitting component to provide energy to the energypropagation layer.

In another example of the second aspect and the above examples, theapparatus can further include a separator structure extending from thedetector structure toward the transparent layer.

In a further example of the second aspect and the above examples, theapparatus can further include a filter layer disposed between the devicestructure and the transparent layer, wherein the filter layer isconfigured to permit the transmission of a wavelength spectrumassociated with a dye.

In an additional example of the second aspect and the above examples,the apparatus can further include a well structure defining wellsdisposed on the transparent layer opposite the detector structure.

In another example of the second aspect and the above examples, theapparatus further includes a pad structure disposed on the transparentlayer opposite the detector structure.

In a further example of the second aspect and the above examples, theapparatus further includes a lid, the flow volume defined between thelid and the transparent layer.

In an additional example of the second aspect and the above examples,the transparent layer comprises an electrode.

In a third aspect, an apparatus includes a transparent layer defining asurface exposed to a flow volume and to secure a target polynucleotide;a well structure defining a plurality of wells that expose thetransparent layer; an energy propagation layer disposed opposite thesurface of the transparent layer and the well structure; an excitationfilter layer secured to the energy propagation layer opposite thetransparent layer; a color filter layer secured to the excitation filterlayer opposite the energy propagation layer; a detector structuresecured to the color filter layer opposite the excitation filter layer,the detector structure defining a plurality of pixels, each pixelincluding at least two detectors, each pixel of the plurality of pixelsuniquely optically associated with a well of the plurality of wells; anda plurality of opaque structures disposed between pixels and extendingfrom the detector structure toward the transparent layer.

In a fourth aspect, a system includes a fluidics system including avalve structure and a plurality of reagent containers; a computationalsystem including a controller; and an integrated device. The integrateddevice includes a transparent layer defining a surface exposed to a flowvolume and to secure a target polynucleotide; and a detector structuresecured to the transparent layer and including a plurality of detectorsto detect a fluorescent signal emitted during nucleotide incorporationusing the target polynucleotide as a template, wherein the fluidicssystem is in communication with the flow volume, and wherein thecontroller is in communication with the detector structure.

In a fifth aspect, a method of obtaining sequence information from apolynucleotide target includes applying a sequencing device to asequencing system, the sequencing device defining a flow volume andincluding a transparent layer including a surface exposed to the flowvolume and a detector structure disposed on an opposite side of thetransparent layer from the flow volume; applying a polynucleotide to thesequencing device; incorporating at least one nucleotide into a nascentnucleic acid molecule using the polynucleotide as a template; anddetecting a fluorescent signal with the detector structure, thefluorescent signal indicative of nucleotide incorporation.

In an example of the fifth aspect, the nucleotide solution includes atleast two dye modified nucleotide types.

In another example of the fifth aspect or the above examples, thenucleotide solution includes four different dye modified nucleotidetypes.

In a further example of the fifth aspect or the above examples, applyingthe nucleotide solution includes sequentially applying at least twodifferent nucleotide solutions.

In a sixth aspect, a method for sequencing a target polynucleotideincludes contacting the device of any one of the first, second, or thirdaspects or examples thereof with an enzymatic reaction, wherein theenzymatic reaction includes contacting: (i) a polymerase with (ii) atleast one target polynucleotide which is base paired with a primer andwith (iii) at least one type of a nucleotide having an opticallydetectable moiety, thereby incorporating a nucleotide onto the primer.

In an example of the sixth aspect, the method further includes a)generating an optically detectable signal by exciting the opticallydetectable moiety with an excitation source; and b) detecting theoptically detectable signal.

In another example of the sixth aspect and the above examples, themethod further includes identifying the incorporated nucleotide.

In a further example of the sixth aspect and the above examples, themethod further includes contacting the device with a second enzymaticreaction, wherein the second enzymatic reaction includes contacting: (i)a second polymerase with (ii) the at least one target polynucleotidewhich is base paired with the primer and with (iii) at least one type ofa nucleotide having an optically detectable moiety, therebyincorporating a second nucleotide onto the primer.

In an additional example of the sixth aspect and the above examples, themethod further includes a) generating a second optically detectablesignal by exciting the second optically detectable moiety with anexcitation source; and b) detecting the second optically detectablesignal.

In another example of the sixth aspect and the above examples, themethod further includes identifying the second incorporated nucleotide.

In a further example of the sixth aspect and the above examples, themethod further includes contacting the device with a second enzymaticreaction, wherein the second enzymatic reaction includes contacting: (i)a second polymerase with (ii) the at least one target polynucleotidewhich is base paired with the primer and with (iii) at least one type ofa nucleotide lacking an optically detectable moiety, therebyincorporating onto the primer the at least one type of nucleotidelacking an optically detectable moiety. For example, the method furtherincludes a) contacting the device with a third enzymatic reaction, whichincludes at least one type of a second nucleotide lacking an opticallydetectable moiety; and b) incorporating onto the primer the secondnucleotide lacking the optically detectable moiety.

In an additional example of the sixth aspect and the above examples, themethod further includes a) repeating the step of contacting the devicewith a third enzymatic reaction, wherein the enzymatic reaction includesat least one type of a nucleotide having an optically detectable moiety;b) generating a second optically detectable signal by exciting theoptically detectable moiety with an excitation source; c) detecting thesecond optically detectable signal; and d) identifying the secondincorporated nucleotide.

In another example of the sixth aspect and the above examples, themethod further includes a) contacting the device with a third enzymaticreaction, wherein the enzymatic reaction includes at least one type of anucleotide having an optically detectable moiety; b) generating a secondoptically detectable signal by exciting the optically detectable moietywith an excitation source; c) detecting the second optically detectablesignal; and d) identifying the second incorporated nucleotide.

In a seventh aspect, a method for generating an energy transfer signalincludes contacting the above devices with a enzymatic reaction, whereinthe enzymatic reaction includes contacting: (i) a mutant polymerasehaving altered nucleotide incorporation kinetics and linked to an energytransfer donor moiety with (ii) a target polynucleotide which is basepaired with a primer and with (iii) at least one type of a nucleotidehaving an energy transfer acceptor moiety, thereby incorporating anucleotide onto the primer, and locating the polymerase and the at leastone type of nucleotide in close proximity with each other to generatethe energy transfer signal, wherein the altered nucleotide incorporationkinetics includes altered polymerase binding to the target molecule,altered polymerase binding to the nucleotide, altered polymerasecatalyzing nucleotide incorporation, altered the polymerase cleaving thephosphate group or substituted phosphate group, or altered polymerasereleasing the cleavage product.

In an example of the sixth and seventh aspects, the associated examples,and the above examples, the first polymerase and the second polymeraseare the same type or different types of polymerases.

In another example of the sixth and seventh aspects, the associatedexamples, and the above examples, the second polymerase and the thirdpolymerase are the same type or different types of polymerases. Forexample, the third polymerase and the fourth polymerase are the sametype or different types of polymerases.

In a further example of the sixth and seventh aspects, the associatedexamples, and the above examples, the first, second, third or fourthpolymerase is a DNA-dependent polymerase, RNA-dependent polymerase, orreverse transcriptase. For example, the first, second, third or fourthpolymerase is a mutant polymerase. In another example, the first,second, third or fourth polymerase has altered nucleotide incorporationkinetics. For example, the altered nucleotide incorporation kineticsincludes altered polymerase binding to the target molecule, alteredpolymerase binding to the nucleotide, altered polymerase catalyzingnucleotide incorporation, altered the polymerase cleaving the phosphategroup or substituted phosphate group, or altered polymerase releasingthe cleavage product.

In an additional example of the sixth and seventh aspects, theassociated examples, and the above examples, the first, second, third orfourth polymerase is linked to an energy transfer donor moiety. Forexample, the energy transfer donor moiety is a nanoparticle or afluorescent dye. In a particular example, the nanoparticle is aninorganic fluorescent nanoparticle.

In another example of the sixth and seventh aspects, the associatedexamples, and the above examples, the at least one target polynucleotideis a single nucleic acid molecule or a plurality of targetpolynucleotides. For example, the plurality of target polynucleotidescomprises a population of nucleic acids having different sequences orhaving substantially identical sequences.

In a further example of the sixth and seventh aspects, the associatedexamples, and the above examples, the at least one target polynucleotideis RNA or DNA.

In an additional example of the sixth and seventh aspects, theassociated examples, and the above examples, the at least one targetpolynucleotide or the primer is attached to the device. For example, theat least one target polynucleotide or the primer is attached thetransparent layer.

In another example of the sixth and seventh aspects, the associatedexamples, and the above examples, the at least one target polynucleotideor the primer is attached to a support. For example, the support is aparticle or microsphere. In another example, the plurality of nucleicacid molecules have different sequences or have substantially identicalsequences.

In a further example of the sixth and seventh aspects, the associatedexamples, and the above examples, the at least one type of nucleotidecomprises one type of nucleotide, or no more than two different types ofnucleotides, or no more than three different types of nucleotides, or nomore than four different types of nucleotides.

In an additional example of the sixth and seventh aspects, theassociated examples, and the above examples, the at least one type ofsecond nucleotide comprises one type of nucleotide, or no more than twodifferent types of nucleotides, or no more than three different types ofnucleotides, or no more than four different types of nucleotides.

In another example of the sixth and seventh aspects, the associatedexamples, and the above examples, the at least one type of nucleotide isadenosine, guanosine, cytosine, thymidine, uridine or inosine.

In a further example of the sixth and seventh aspects, the associatedexamples, and the above examples, the at least one type of nucleotide isadenosine, guanosine, cytosine, thymidine, uridine or inosine.

In an additional example of the sixth and seventh aspects, theassociated examples, and the above examples, the at least one type ofnucleotide comprises 3-10 or more phosphate groups.

In another example of the sixth and seventh aspects, the associatedexamples, and the above examples, the at least one type of nucleotidecomprises 3-10 or more phosphate groups. For example, the differenttypes of nucleotides differ from each other in their base, sugar orphosphate backbone.

In a further example of the sixth and seventh aspects, the associatedexamples, and the above examples, the at least one type of nucleotidecomprises a non-incorporatable nucleotide.

In an additional example of the sixth and seventh aspects, theassociated examples, and the above examples, the optically detectablemoiety is linked to any one of the phosphate groups, the base or thesugar of the at least one type of nucleotide.

In another example of the sixth and seventh aspects, the associatedexamples, and the above examples, the optically detectable moiety islinked to the nucleotide via a linker that is cleavable with light, achemical compound or an enzyme. For example, the method further includesremoving the optically detectable moiety by cleaving the cleavablelinker with light, a chemical compound or an enzyme.

In a further example of the sixth and seventh aspects, the associatedexamples, and the above examples, the optically detectable moiety is afluorophore.

In an additional example of the sixth and seventh aspects, theassociated examples, and the above examples, the optically detectablesignal comprises a fluorescent signal.

In another example of the sixth and seventh aspects, the associatedexamples, and the above examples, the at least one type of a nucleotideis linked to an optically detectable moiety comprising an energytransfer acceptor moiety.

In a further example of the sixth and seventh aspects, the associatedexamples, and the above examples, the at least one type of a nucleotideis linked to an energy transfer acceptor moiety and the first, second orthird polymerase is linked to an energy transfer donor moiety, andincorporating the at least one type of nucleotide onto the primer,brings the first, second or third polymerase and the at least one typeof a nucleotide into close proximity with each other to generate anenergy transfer signal. For example, the energy transfer signalcomprises a fluorescent signal.

In an additional example of the sixth and seventh aspects, theassociated examples, and the above examples, the at least one type ofnucleotide is linked to at least one inhibitor moiety that inhibitsincorporation of a subsequent nucleotide. For example, the inhibitormoiety is linked to the nucleotide at any position of the base, or anyposition of the sugar. In another example, the inhibitor moiety islinked to the nucleotide via a linker that is cleavable with light, achemical compound or an enzyme. For example, the method further includesremoving the inhibitor moiety by cleaving the cleavable linker withlight, a chemical compound or an enzyme.

In another example of the sixth and seventh aspects, the associatedexamples, and the above examples, the at least one type of a nucleotidehaving an optically detectable moiety comprises a mixture of differenttypes of nucleotides containing at least one type of a nucleotide havingan optically detectable moiety and at least one type of nucleotidelacking an optically detectable moiety. For example, the mixture ofdifferent types of nucleotides includes two, three, four or moredifferent types of nucleotides. In an example, the different nucleotidesin the mixture of nucleotides are contacted sequentially with the first,second or third polymerase. For example, the different nucleotides inthe mixture of nucleotides are contacted essentially simultaneously withthe first, second or third polymerase. In a further example, the mixtureof different types of nucleotides includes: (iv) one type of nucleotidehaving an optically detectable moiety and two or three different typesof nucleotides that lack an optically detectable moiety; or (v) twodifferent types of nucleotides having different optically detectablemoieties and one or two different types of nucleotides that lack anoptically detectable moiety; or (vi) three different types ofnucleotides having different optically detectable moieties and one typeof nucleotide that lacks an optically detectable moiety. In anotherexample, the mixture of different types of nucleotides includes anon-incorporatable nucleotide.

In a further example of the sixth and seventh aspects, the associatedexamples, and the above examples, the excitation source iselectromagnetic energy. For example, the electromagnetic energy islight.

In an additional example of the sixth and seventh aspects, theassociated examples, and the above examples, the device comprises aplurality of sites on the device arranged in an organized or randomarray.

In another example of the sixth and seventh aspects, the associatedexamples, and the above examples, the device comprises a plurality ofsites on the device arranged in a rectilinear or hexagonal pattern.

In a further example of the sixth and seventh aspects, the associatedexamples, and the above examples, the method further includes subjectinga plurality of target polynucleotides to the same enzymatic reaction inparallel, wherein the plurality of target polynucleotides comprises apopulation of nucleic acids having substantially identical sequences ordifferent sequences.

In an example of the sixth and seventh aspects, the associated examples,and the above examples, the target polynucleotide comprises a singlenucleic acid molecule.

In an eighth aspect, a method for sequencing a target polynucleotideincludes contacting the device of any one of the first, second, or thirdaspects with an enzymatic reaction, wherein the enzymatic reactionincludes: contacting at least one type of a labeled nucleotide to acomplex having a first polymerase bound to at least one targetpolynucleotide that is bound to a primer, under suitable conditions totransiently-bind, without polymerizing, the at least one type of labelednucleotide to the polymerase in a nucleic acid template-dependentmanner; detecting the transiently-bound labeled nucleotide; andidentifying the labeled nucleotide transiently-bound to the polymerase,

In an example of the eighth aspect, the method further includes removingthe transiently-bound nucleotide; and contacting the complex with atleast one type of a second nucleotide under suitable conditions for asecond polymerase to polymerize the nucleotide.

In another example of the eighth aspect and the above examples, the atleast one target polynucleotide comprises a single nucleic acidmolecule.

In a further example of the eighth aspect and the above examples, thesuitable conditions in the first step include: (i) reducing the levelsor omission of a cation that permits nucleotide incorporation oraddition of a cation that inhibits nucleotide incorporation; (ii) thepolymerase selectively binds the nucleotide in a template-dependentmanner and exhibits reduced nucleotide incorporation activity; (iii) theat least one type of labeled nucleotide is a labeled non-incorporatablenucleotide; or (iv) the primer includes a non-extendible polymerizationinitiation site.

In an additional example of the eighth aspect and the above examples,the suitable conditions in step (a) comprise: (i) cations present at aconcentration that inhibits nucleotide incorporation; (ii) thepolymerase selectively binds the nucleotide in a template-dependentmanner and exhibits reduced nucleotide incorporation activity; (iii) theat least one type of labeled nucleotide is a labeled non-incorporatablenucleotide; or (iv) the primer includes a non-extendible polymerizationinitiation site. For example, the cation that inhibits nucleotideincorporation is calcium, scandium, titanium, vanadium, chromium, iron,cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium,rhodium, or strontium.

In a ninth aspect, a method for nucleic acid sequencing includescontacting the device of any one of the first, second, and third aspectswith an enzymatic reaction, wherein the enzymatic reaction includes:transiently binding, without polymerizing, at least a first type of alabeled nucleotide to a first polymerase in the presence of a cationthat inhibits nucleotide incorporation by the polymerase, wherein thefirst polymerase is bound to at least one target polynucleotide that isbound to a primer, and wherein the first type of nucleotide includes anoptically detectable moiety; detecting the first type oftransiently-bound nucleotide; and identifying the first type oftransiently-bound nucleotide,

In an example of the ninth aspect, the method further includes removingthe transiently-bound first type of nucleotide; and contacting thecomplex with at least one type of a second nucleotide under suitableconditions for a second polymerase to polymerize the second type ofnucleotide in a template-dependent manner.

In another example of the ninth aspect and the above examples, the atleast one target polynucleotide comprises a single nucleic acidmolecule.

In a further example of the ninth aspect and the above examples, thesuitable conditions in the second step further include any one or moreof the following: (i) including a cation that permits nucleotideincorporation or reducing the levels or omission of a cation thatinhibits nucleotide incorporation; (ii) the first polymerase selectivelybinds the second type of nucleotide in a template-dependent manner andpolymerizes the bound second type of nucleotide; (iii) the second typeof nucleotide is an incorporatable nucleotide; and (iv) the primer is anextendible polymerization initiation site. For example, the cation thatinhibits nucleotide incorporation is calcium, scandium, titanium,vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium,germanium, arsenic, selenium, rhodium, or strontium.

In an additional example of the ninth aspect and the above examples, thefirst and second polymerases are the same polymerase, or differentpolymerases of the same type, or different types of polymerases.

In another example of the ninth aspect and the above examples, the firstor second polymerase is a DNA-dependent polymerase, RNA-dependentpolymerase, or reverse transcriptase.

In a further example of the ninth aspect and the above examples, thefirst or second polymerase is a mutant polymerase.

In an additional example of the ninth aspect and the above examples, thefirst or second polymerase has altered nucleotide incorporationkinetics. For example, the altered nucleotide incorporation kineticsincludes altered polymerase binding to the target molecule, alteredpolymerase binding to the nucleotide, altered polymerase catalyzingnucleotide incorporation, altered the polymerase cleaving the phosphategroup or substituted phosphate group, or altered polymerase releasingthe cleavage product.

In another example of the ninth aspect and the above examples, the firstor second polymerase comprises an RB69 polymerase, phi29 polymerase,B103 polymerase, or Klenow fragment polymerase.

In a further example of the ninth aspect and the above examples, thefirst or second polymerase is linked to an energy transfer donor moiety.For example, the energy transfer donor moiety is a nanoparticle or afluorescent dye. In an example, the nanoparticle is an inorganicfluorescent nanoparticle.

In an additional example of the ninth aspect and the above examples, theat least one target polynucleotide is a single nucleic acid molecule ora plurality of target polynucleotides. For example, the plurality oftarget polynucleotides comprises a population of nucleic acids havingdifferent sequences or having substantially identical sequences.

In another example of the ninth aspect and the above examples, the atleast one target polynucleotide is RNA or DNA.

In a further example of the ninth aspect and the above examples, the atleast one target polynucleotide or the primer is attached to the device.For example, the at least one target polynucleotide or the primer isattached the transparent layer of the device.

In an additional example of the ninth aspect and the above examples, theat least one target polynucleotide or the primer is attached to asupport.

In another example of the ninth aspect and the above examples, thesupport is a particle or microsphere.

In a further example of the ninth aspect and the above examples, thelabeled nucleotide comprises one type of nucleotide, or no more than twodifferent types of nucleotides, or no more than three different types ofnucleotides, or no more than four different types of nucleotides.

In an additional example of the ninth aspect and the above examples, theat least one type of second nucleotide comprises one type of nucleotide,or no more than two different types of nucleotides, or no more thanthree different types of nucleotides, or no more than four differenttypes of nucleotides.

In another example of the ninth aspect and the above examples, thelabeled nucleotide is adenosine, guanosine, cytosine, thymidine, uridineor inosine.

In a further example of the ninth aspect and the above examples, atleast one type of a second nucleotide is adenosine, guanosine, cytosine,thymidine, uridine or inosine.

In an additional example of the ninth aspect and the above examples, thelabeled nucleotide comprises 3-10 or more phosphate groups.

In another example of the ninth aspect and the above examples, at leastone type of a second nucleotide comprises 3-10 or more phosphate groups.

In a further example of the ninth aspect and the above examples, thenucleotide comprises a non-incorporatable nucleotide.

In an additional example of the ninth aspect and the above examples, thelabeled nucleotide is linked to at least one optically detectablemoiety.

In another example of the ninth aspect and the above examples, the atleast one type of a second nucleotide is linked to at least oneoptically detectable moiety

In a further example of the ninth aspect and the above examples, the atleast one optically detectable moiety is linked to any one of thephosphate groups, the base or the sugar of the nucleotide.

In an additional example of the ninth aspect and the above examples, theoptically detectable moiety is linked to the nucleotide via a linkerthat is cleavable with light, a chemical compound or an enzyme. Forexample, the optically detectable moiety is a fluorophore. In anotherexample, the optically detectable moiety emits a fluorescent signal. Ina further example, the optically detectable moiety comprises an energytransfer acceptor moiety.

In another example of the ninth aspect and the above examples, thelabeled nucleotide is linked to an energy transfer acceptor moiety andthe first polymerase is linked to an energy transfer donor moiety, andthe transient binding the nucleotide to the polymerase brings the firstpolymerase and the labeled nucleotide in close proximity with each otherto generate an energy transfer signal. For example, the energy transfersignal comprises a fluorescent signal.

In a further example of the ninth aspect and the above examples, thelabeled nucleotide is linked to at least one inhibitor moiety thatinhibits incorporation of a subsequent nucleotide. For example, theinhibitor moiety is linked to the nucleotide at any position of the baseor the sugar. In an example, the inhibitor moiety is linked to thenucleotide via a linker that is cleavable with light, a chemicalcompound or an enzyme.

In an additional example of the ninth aspect and the above examples, thelabeled nucleotide comprises a mixture of different types of nucleotidescontaining at least one type of a nucleotide having an opticallydetectable moiety and at least one type of nucleotide lacking anoptically detectable moiety. For example, the mixture of different typesof nucleotides includes two, three, four or more different types ofnucleotides. In an example, the different nucleotides in the mixture ofnucleotides are contacted sequentially with the first polymerase. Inanother example, the different nucleotides in the mixture of nucleotidesare contacted essentially simultaneously with the first polymerase. Inan additional example, the mixture of different types of nucleotidesincludes: (iv) one type of nucleotide having an optically detectablemoiety and two or three different types of nucleotides that lack anoptically detectable moiety; or (v) two different types of nucleotideshaving different optically detectable moieties and one or two differenttypes of nucleotides that lack an optically detectable moiety; or (vi)three different types of nucleotides having different opticallydetectable moieties and one type of nucleotide that lacks an opticallydetectable moiety. In a further example, the mixture of different typesof nucleotides includes a non-incorporatable nucleotide.

In another example of the ninth aspect and the above examples, themethod further includes a) generating an optically detectable signal byexciting the optically detectable moiety with an excitation source; andb) detecting the optically detectable signal. For example, theexcitation source is electromagnetic energy. In an example, theelectromagnetic energy is light.

In a further example of the ninth aspect and the above examples, thedevice comprises a plurality of sites on the device arranged in anorganized or random array.

In an additional example of the ninth aspect and the above examples, thedevice comprises a plurality of sites on the device arranged in arectilinear or hexagonal pattern.

In another example of the ninth aspect and the above examples, themethod further includes subjecting a plurality of target polynucleotidesto the same enzymatic reaction in parallel, wherein the plurality oftarget polynucleotides comprises a population of nucleic acids havingsubstantially identical sequences or different sequences.

In an tenth aspect, a method for sequencing a target polynucleotideincludes contacting the device of any one of the first, second or thirdaspects with an enzymatic reaction, wherein the enzymatic reactionincludes: forming a reaction mixture containing a polymerase and atarget polynucleotide, wherein the reaction mixture includes aconcentration of cation at which nucleotide polymerization by thepolymerase is inhibited; contacting, within the reaction mixture, afirst type of nucleotide containing a detectable moiety with thepolymerase and transiently binding the first type of nucleotide to thepolymerase in a template-dependent manner without polymerizing thetransiently bound nucleotide at the primer by the polymerase; detectinga signal generated by the detectable moiety while the first type ofnucleotide is transiently bound to the polymerase; removing thetransiently bound nucleotide from the polymerase; and contacting thepolymerase with a second type of nucleotide and polymerizing the secondtype of nucleotide at the primer using the polymerase.

In an example of the tenth aspect, wherein the forming in the first stepincludes any one or more of the following: (i) reducing the levels oromission of a cation that permits nucleotide incorporation or additionof a cation that inhibits nucleotide incorporation.

In another example of the tenth aspect and the above examples, thesuitable conditions in step comprise any one or more of the following:(i) including a cation that permits nucleotide incorporation or reducingthe levels or omission of a cation that inhibits nucleotideincorporation; (ii) using a first polymerase which selectively binds thesecond type of nucleotide in a template-dependent manner and polymerizesthe bound second type of nucleotide; (iii) using a second type ofnucleotide which includes an incorporatable nucleotide; or (iv) using aprimer having an extendible polymerization initiation site

In a further example of the tenth aspect and the above examples, thecation that inhibits nucleotide incorporation is calcium, scandium,titanium, vanadium, chromium, iron, cobalt, nickel, copper, zinc,gallium, germanium, arsenic, selenium, rhodium, or strontium.

In an additional example of the tenth aspect and the above examples, thetransiently binding in step (b) includes contacting the polymerase witha sufficient amount of a divalent cation to inhibit nucleotideincorporation by the first polymerase. For example, the divalent cationincludes calcium.

In another example of the tenth aspect and the above examples, themethod further includes repeating the first three steps at least once.

In a further example of the tenth aspect and the above examples, themethod includes repeating the fourth and fifth steps at least once.

Note that not all of the activities described above in the generaldescription or the examples are required, that a portion of a specificactivity may not be required, and that one or more further activitiesmay be performed in addition to those described. Still further, theorder in which activities are listed are not necessarily the order inwhich they are performed.

In the foregoing specification, the concepts have been described withreference to specific embodiments. However, one of ordinary skill in theart appreciates that various modifications and changes can be madewithout departing from the scope of the invention as set forth in theclaims below. Accordingly, the specification and figures are to beregarded in an illustrative rather than a restrictive sense, and allsuch modifications are intended to be included within the scope ofinvention.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having” or any other variation thereof, areintended to cover a non-exclusive inclusion. For example, a process,method, article, or apparatus that comprises a list of features is notnecessarily limited only to those features but may include otherfeatures not expressly listed or inherent to such process, method,article, or apparatus. Further, unless expressly stated to the contrary,“or” refers to an inclusive-or and not to an exclusive-or. For example,a condition A or B is satisfied by any one of the following: A is true(or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

Also, the use of “a” or “an” are employed to describe elements andcomponents described herein. This is done merely for convenience and togive a general sense of the scope of the invention. This descriptionshould be read to include one or at least one and the singular alsoincludes the plural unless it is obvious that it is meant otherwise.

Benefits, other advantages, and solutions to problems have beendescribed above with regard to specific embodiments. However, thebenefits, advantages, solutions to problems, and any feature(s) that maycause any benefit, advantage, or solution to occur or become morepronounced are not to be construed as a critical, required, or essentialfeature of any or all the claims.

After reading the specification, skilled artisans will appreciate thatcertain features are, for clarity, described herein in the context ofseparate embodiments, may also be provided in combination in a singleembodiment. Conversely, various features that are, for brevity,described in the context of a single embodiment, may also be providedseparately or in any subcombination. Further, references to valuesstated in ranges include each and every value within that range.

What is claimed is:
 1. A device comprising: a transparent layer defininga surface exposed to a flow volume and to secure a target polynucleotidetemplate; and a detector structure in optical communication with andsecured to the transparent layer and including a plurality of detectorsconfigured to detect a fluorescent signal emitted during nucleotideincorporation during template-dependent nucleic acid synthesis, whereinthe detector structure includes a plurality of pixels, each pixel of theplurality of pixels including a set of detectors of the plurality ofdetectors, wherein the set of detectors are defined within thesemiconductor structure by a p-type substrate, a deep n-type implant, ashallow n-type implant, and a p-type implant disposed over the shallown-type implant.
 2. The device of claim 1, wherein each pixel of theplurality of pixels includes at least two detectors of the plurality ofdetectors.
 3. The device of claim 2, where the at least two detectorsare disposed one over the other when viewed in cross-section.
 4. Thedevice of claim 2, wherein each pixel includes at least four detectors.5. The device of claim 1, wherein the transparent layer includes anenergy propagation layer.
 6. The device of claim 1, further comprisingan energy propagation layer disposed between the transparent layer andthe detector structure.
 7. The device of claim 6, wherein the energypropagation layer includes a total internal reflection layer.
 8. Thedevice of claim 6, further comprising an energy emitting component toprovide energy to the energy propagation layer.
 9. The device of claim1, further comprising a separator structure extending from the detectorstructure toward the transparent layer, the separator structure opaqueto the fluorescent signal.
 10. The device of claim 1, further comprisinga filter layer disposed between the device structure and the transparentlayer.
 11. The device of claim 10, wherein the filter layer isconfigured to limit transmission of excitation energy.
 12. The device ofclaim 1, further comprising a well structure defining wells disposed onthe transparent layer opposite the detector structure.
 13. The device ofclaim 1, further comprising a lid, the flow volume defined between thelid and the transparent layer.
 14. A method of obtaining sequenceinformation from a polynucleotide target, the method comprising:applying a sequencing device to a sequencing system, the sequencingdevice defining a flow volume and including a transparent layerincluding a surface exposed to the flow volume and a detector structuredisposed on an opposite side of the transparent layer from the flowvolume, the detector structure including a plurality of detectorsconfigured to detect a fluorescent signal emitted during nucleotideincorporation during template-dependent nucleic acid synthesis, whereinthe detector structure includes a plurality of pixels, each pixel of theplurality of pixels including a set of detectors of the plurality ofdetectors, wherein the set of detectors are defined within thesemiconductor structure by a p-type substrate, a deep n-type implant, ashallow n-type implant, and a p-type implant disposed over the shallown-type implant; applying a polynucleotide to the sequencing device;incorporating at least one dye modified nucleotide into a nascentnucleic acid molecule using the polynucleotide as a template; anddetecting a fluorescent signal with the detector structure from a dye ofthe at least one dye modified nucleotide, the fluorescent signalindicative of nucleotide incorporation of the at least one dye modifiednucleotide.
 15. The method of claim 14, wherein the nucleotide solutionincludes at least two dye modified nucleotide types.
 16. The method ofclaim 14, wherein the nucleotide solution includes four different dyemodified nucleotide types.
 17. The method of claim 14, wherein applyingthe nucleotide solution includes sequentially applying at least twodifferent nucleotide solutions.
 18. The method of claim 14, wherein thetransparent layer includes an energy propagation layer, the methodfurther comprising applying excitation energy to the energy propagationlayer, the excitation energy exiting the dye of the at least one dyemodified nucleotide.
 19. The method of claim 18, wherein the sequencingdevice further includes a filter layer disposed between the devicestructure and the transparent layer, wherein the filter layer isconfigured to limit transmission of excitation energy to the pluralityof detectors.
 20. The method of claim 14, wherein the sequencing devicefurther includes a well structure defining wells disposed on thetransparent layer opposite the detector structure, wherein applying apolynucleotide to the sequencing device includes applying thepolynucleotides into the wells.